Mass Spectrometry - Mass Spec History

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What is Mass Spectrometry?

Basics of Mass Spectrometry

Mass spectrometry has been described as the smallest scale in the w orld, not because of the massspectrometer’s size but because of the size of w hat it w eighs -- molecules. Over the past decade, massspectrometry has undergone tremendous technological improvements allow ing for its application to proteins,peptides, carbohydrates, DNA, drugs, and many other biologically relevant molecules. Due to ionization sourcessuch as electrospray ionization and matrix-assisted laser desorption/ ionization (MALDI), mass spectrometry hasbecome an irreplaceable tool in the biological sciences. This chapter provides an overview of massspectrometry, focusing on ionization sources and their signif icance in the development of mass spectrometry inbiomolecular analysis.

A mass spectrometer determines the mass of a molecule by measuring the mass-to-charge ratio (m/z) of itsion. Ions are generated by inducing either the loss or gain of a charge from a neutral species. Once formed, ionsare electrostatically directed into a mass analyzer w here they are separated according to m/z and finallydetected. The result of molecular ionization, ion separation, and ion detection is a spectrum that can providemolecular mass and even structural information. An analogy can be draw n betw een a mass spectrometer and aprism, as show n in Figure 1.1. In the prism, light is separated into its component w avelengths w hich are thendetected w ith an optical receptor, such as visualization. Similarly, in a mass spectrometer the generated ions areseparated in the mass analyzer, digitized and detected by an ion detector (such as an electron multiplier, Chapter2).

Figure 1.1. The mass analysis process as compared to the dispersion of light by a prism.

So What is Mass Spectrometry?

John B. Fenn, the originator of electrospray ionization for biomolecules and the 2002 Nobel Laureate inChemistry, probably gave the most apt answ er to this question:

Mass spectrometry is the art of measuring atoms and molecules to determine their molecularweight. Such mass or weight information is sometimes sufficient, frequently necessary, andalways useful in determining the identity of a species. To practice this art one puts charge onthe molecules of interest, i.e., the analyte, then measures how the trajectories of the resultingions respond in vacuum to various combinations of electric and magnetic fields.Clearly, the sine qua non of such a method is the conversion of neutral analyte molecules intoions. For small and simple species the ionization is readily carried by gas-phase encountersbetween the neutral molecules and electrons, photons, or other ions. In recent years, theefforts of many investigators have led to new techniques for producing ions of species too largeand complex to be vaporized without substantial, even catastrophic, decomposition.

Some Basics

Four basic components are, for the most part, standard in all mass spectrometers (Figure 1.2): a sample inlet,an ionization source, a mass analyzer and an ion detector. Some instruments combine the sample inlet and theionization source, w hile others combine the mass analyzer and the detector. How ever, all sample moleculesundergo the same processes regardless of instrument configuration. Sample molecules are introduced into theinstrument through a sample inlet. Once inside the instrument, the sample molecules are converted to ions in theionization source, before being electrostatically propelled into the mass analyzer. Ions are then separatedaccording to their m/z w ithin the mass analyzer. The detector converts the ion energy into electrical signals,w hich are then transmitted to a computer.

Sample Introduction Techniques

Sample introduction w as an early challenge in mass spectrometry. In order to perform mass analysis on asample, w hich is initially at atmospheric pressure (760 torr), it must be introduced into the instrument in such aw ay that the vacuum inside the instrument remains relatively unchanged (~10-6 torr). The most common methodsof sample introduction are direct insertion w ith a probe or plate commonly used w ith MALDI-MS, direct infusion orinjection into the ionization source such as ESI-MS.

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Figure 1.2. Components of a mass spectrometer. Note that the ion source does not have to be w ithin thevacuum of the mass spectrometer. For instance, ESI and APCI are at atmospheric pressure and are know n asatmospheric pressure ionization (API) sources.

Direct Insertion: Using an insertion probe/plate (Figure 1.3) is a very simple w ay to introduce a sample into aninstrument. The sample is f irst placed onto a probe and then inserted into the ionization region of the massspectrometer, typically through a vacuum interlock. The sample is then subjected to any number of desorptionprocesses, such as laser desorption or direct heating, to facilitate vaporization and ionization.

Direct Infusion: A simple capillary or a capillary column is used to introduce a sample as a gas or in solution.Direct infusion is also useful because it can eff iciently introduce small quantities of sample into a massspectrometer w ithout compromising the vacuum. Capillary columns are routinely used to interface separationtechniques w ith the ionization source of a mass spectrometer. These techniques, including gas chromatography(GC) and liquid chromatography (LC), also serve to separate a solution’s different components prior to massanalysis. In gas chromatography, separation of different components occurs w ithin a glass capillary column. Asthe vaporized sample exits the gas chromatograph, it is directly introduced into the mass spectrometer.

In the 1980s the incapability of liquid chromatography (LC) w ith mass spectrometry w as due largely to theionization techniques being unable to handle the continuous flow of LC. How ever, electrospray ionization (ESI),atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) now allow sLC/MS to be performed routinely (Figure 1.4).

Figure 1.3. Samples are often introduced into the mass spectrometer using a direct insertion probe, a capillarycolumn (EI w ith GC/MS or ESI) or a sample plate (MALDI). The vacuum interlock allow s for the vacuum of themass spectrometer to be maintained w hile the instrument is not in use. It also allow s for the sample (atatmospheric pressure) to be introduced into the high vacuum of the mass spectrometer.

Figure 1.4. Interfacing liquid chromatography w ith electrospray ionization mass spectrometry. Liquidchromatography/mass spectrometry (LC/MS) ion chromatogram and the corresponding electrospray massspectrum are show n. Gas chromatography mass spectrometry (GC/MS) produces results in much the same w ayas LC/MS, how ever, GC/MS uses an electron ionization source, w hich is limited by thermal vaporization (UVrefers to ultraviolet and TIC is the total ion current).

( Back to the Table of Contents )

Ionization

Ionization method refers to the mechanism of ionization w hile the ionization source is the mechanical devicethat allow s ionization to occur. The different ionization methods, summarized here, w ork by either ionizing aneutral molecule through electron ejection, electron capture, protonation, cationization, or deprotonation, or bytransferring a charged molecule from a condensed phase to the gas phase.

Protonation

Scheme 1.1. An example of a massspectrum obtained via protonation

Protonation is a method ofionization by w hich a proton isadded to a molecule, producinga net positive charge of 1+ forevery proton added. Positivecharges tend to reside on themore basic residues of themolecule, such as amines, toform stable cations. Peptidesare often ionized viaprotonation. Protonation can beachieved via matrix-assistedlaser desorption/-ionization(MALDI), electrospray ionization(ESI) and atmospheric pressurechemical ionization (APCI).

Deprotonation

Scheme 1.2. An example of a massspectrum of sialic acid obtained viadeprotonation.

Deprotonation is an ionizationmethod by w hich the netnegative charge of 1- isachieved through the removalof a proton from a molecule.This mechanism of ion-ization,commonly achieved via MALDI,ESI, and APCI is very useful foracidic species includingphenols, carboxylic acids, andsulfonic acids. The negative ionmass spectrum of sialic acid isshow n in Scheme 1.2.

Cationization

Scheme 1.3. An example of a massspectrum obtained via cationization.

M + Cation+ → MCation+

Cationization is a method ofionization that produces acharged complex by non-covalently adding a positivelycharged ion to a neutralmolecule. While protonationcould fall under this samedefinition, cationization isdistinct for its addition of acation adduct other than aproton (e.g. alkali, ammonium).Moreover, it is know n to beuseful w ith molecules unstableto protonation. The binding ofcations other than protons to amolecule is naturally lesscovalent, therefore, the chargeremains localized on the cation.

This minimizes delocalization ofthe charge and fragmentation ofthe molecule. Cationization iscommonly achieved via MALDI,ESI, and APCI. Carbohydrates

f

are excellent candidates for thisionization mechanism, w ith Na+

a common cation adduct.

Transfer of a charged molecule to the gas phase

Scheme 1.4. An example of a massspectrum of tetraphenylphosphine obtainedvia transfer of a charged species fromsolution into the gas phase.

The transfer of compoundsalready charged in solution isnormally achieved through thedesorption or ejection of thecharged species from thecondensed phase into the gasphase. This transfer iscommonly achieved via MALDIor ESI. The positive ion massspectrum oftetraphenylphosphine is show nin Scheme 1.4.

Electron ejection

Scheme 1.5. An example of a massspectrum obtained via electron ejection.

As its name implies, electronejection achieves ionizationthrough the ejection of anelectron to produce a 1+ netpositive charge, often formingradical cations. Observed mostcommonly w ith electronionization (EI) sources, electronejection is usually performed onrelatively nonpolar compoundsw ith low molecular w eights andit is also know n to generatesignif icant fragment ions. Themass spectrum resulting fromelectron ejection of anthraceneis show n in Scheme 1.5.

Electron capture

Scheme 1.6. An example of a massspectrum obtained via electron capture.Electron capture is commonly achieved viaelectron ionization (EI).

With the electron captureionization method, a netnegative charge of 1- isachieved w ith the absorption orcapture of an electron. It is amechanism of ion-izationprimarily observed for

molecules w ith a high electronaff inity, such as halogenatedcompounds. The electroncapture mass spectrum ofhexachloro-benzene is show nin Scheme 1.6.

Table 1.1. Ionization methods, advantages and disadvantages.

IonizationMethod Advantages Disadvantages

Protonation(positive ions) many compounds w ill accept a

proton to become ionizedmany ionization sources such asESI, APCI, FAB, CI and MALDI w illgenerate these species

some compounds are not stableto protonation (i.e. carbohydrates)or cannot accept a proton easily(i.e. hydrocarbons)

Cationization(positive ions) many compounds w ill accept a

cation, such as Na+ or K+ tobecome ionizedmany ionization sources such asESI, APCI, FAB and MALDI w illgenerate these species

tandem mass spectrometryexperiments on cationizedmolecules w ill often generatelimited or no fragmentationinformation

Deprotonation(negative ions) most useful for compounds that

are somew hat acidicmany ionization sources such asESI, APCI, FAB and MALDI w illgenerate these species

compound specif ic

Transfer ofcharged molecule togas phase(positive ornegative ions)

useful w hen the compound isalready chargedmany ionization sources such asESI, APCI, FAB and MALDI w illgenerate these species

only for precharged ions

Electron ejection(positive ions) observed w ith electron ionization

and can provide molecular massas w ell as fragmentationinformation

often generates too muchfragmentationit can be unclear w hether thehighest mass ion is the molecularion or a fragment

Electron capture(negative ions) observed w ith electron ionization

and can provide molecular massas w ell as fragmentationinformation

often generates too muchfragmentationit can be unclear w hether thehighest mass ion is the molecularion or a fragment


Ionization Sources

Prior to the 1980s, electron ionization (EI) w as the primary ionization source for mass analysis. How ever, EIlimited chemists and biochemists to small molecules w ell below the mass range of common bio-organiccompounds. This limitation motivated scientists such as John B. Fenn, Koichi Tanaka, Franz Hillenkamp, Michael

Karas, Graham Cooks, and Michael Barber to develop the new generation of ionization techniques, including fastatom/ion bombardment (FAB), matrix-assisted laser desorption/ionization (MALDI), and electrospray ionization(ESI) (Table 1.2). These techniques have revolutionized biomolecular analyses, especially for large molecules.Among them, ESI and MALDI have clearly evolved to be the methods of choice w hen it comes to biomolecularanalysis.

Table 1.2

Ionization Source Acronym Event

Electrospray ionization ESI evaporation of charged droplets

Nanoelectrospray ionization nanoESI evaporation of charged droplets

Atmospheric pressure chemical ionization APCI corona discharge and proton transfer

Matrix-assisted laser desorption/ionization MALDI photon absorption/proton transfer

Desorption/ionization on silicon DIOS photon absorption/proton transfer

Fast atom/ion bombardment FAB ion desorption/proton transfer

Electron ionization EI electron beam/electron transfer

Chemical ionization CI proton transfer

MALDI and ESI are now the most common ionization sources for biomolecular mass spectrometry, offeringexcellent mass range and sensitivity (Figure 1.5). The follow ing section w ill focus on the principles of ionizationsources, providing some details on the practical aspects of their use as w ell as ionization mechanisms.

Figure 1.5. A glance at the typical sensitivity and mass ranges allow ed by different ionization techniquesprovides a clear answ er to the question of w hich are most useful; electron ionization (EI), atmospheric pressurechemical ionization (APCI) and desorption/ionization on silicon (DIOS) are somew hat limiting in terms of uppermass range, w hile electrospray ionization (ESI), nanoelectrospray ionization (nanoESI), and matrix-assisted laserdesorption ionization (MALDI) have a high practical mass range.

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Electrospray Ionization

The idea of electrospray, w hile not new , has been rejuvenated w ith its recent application to biomolecules. Thefirst electrospray experiments w ere carried out by Chapman in the late 1930s and the practical development ofelectrospray ionization for mass spectrometry w as accomplished by Dole in the late 1960s. Dole also discoveredthe important phenomenon of multiple charging of molecules. It w as Fenn’s w ork that ultimately led to the modernday technique of electrospray ionization mass spectrometry and its application to biological macromolecules.

A more physical explanation of ESI is that the needle voltage produces an electrical gradient on the fluid w hichseparates the charges at the surface. This forces the liquid to emerge from the needle as a Taylor cone. The tipof the Taylor cone protrudes as a f ilament until the liquid reaches the Rayleigh limit w here the surface tensionand electrostatic repulsion are equal and the highly charged droplets leave the f ilament. The droplets that break

aw ay from the filament are attracted to the entrance of the mass spectrometer due to the high opposite voltageat the mass analyzer's entrance. As the droplet moves tow ards the analyzers, the Coulombic repulsion on thesurface exceeds the surface tension, the droplet explodes into smaller droplets ultimately releasing ions.

Figure 1.6. Electrospray ionization (ESI) mass spectrometry.

Electrospray ionization (ESI) is a method routinely used w ith peptides, proteins, carbohydrates, smalloligonucleotides, synthetic polymers, and lipids. ESI produces gaseous ionized molecules directly from a liquidsolution. It operates by creating a f ine spray of highly charged droplets in the presence of an electric f ield. (Anillustration of the electrospray ionization process is show n in Figures 1.6 and 1.7). The sample solution issprayed from a region of the strong electric f ield at the tip of a metal nozzle maintained at a potential of anyw herefrom 700 V to 5000 V. The nozzle (or needle) to w hich the potential is applied serves to disperse the solution intoa fine spray of charged droplets. Either dry gas, heat, or both are applied to the droplets at atmospheric pressurethus causing the solvent to evaporate from each droplet. As the size of the charged droplet decreases, thecharge density on its surface increases. The mutual Coulombic repulsion betw een like charges on this surfacebecomes so great that it exceeds the forces of surface tension, and ions are ejected from the droplet through a“Taylor cone” Figure 1.7. Another possibility is that the droplet explodes releasing the ions. In either case, theemerging ions are directed into an orif ice through electrostatic lenses leading to the vacuum of the massanalyzer. Because ESI involves the continuous introduction of solution, it is suitable for using as an interface w ithHPLC or capillary electrophoresis.

Figure 1.7. and negative ESI of an oligonucleotide (top) and a protein (bottom).

Electrospray ionization is conducive to the formation of singly charged small molecules, but is also w ell-know nfor producing multiply charged species of larger molecules. This is an important phenomenon because the massspectrometer measures the mass-to-charge ratio (m/z) and therefore multiple charging makes it possible toobserve very large molecules w ith an instrument having a relatively small mass range. Fortunately, the softw areavailable w ith all electrospray mass spectrometers facilitates the molecular w eight calculations necessary todetermine the actual mass of the multiply-charged species. Figures 1.8 and 1.9 illustrate the dif ferent chargestates on tw o dif ferent proteins, w here each of the peaks in the mass spectra can be associated w ith dif ferentcharge states of the molecular ion. Multiple charging has other important advantages in tandem massspectrometry. One advantage is that upon fragmentation you observe more fragment ions w ith multiply chargedprecursor ions than w ith singly charged precursor ions.

Multiple charging: A 10,000 Da protein and its theoretical mass spectrum w ith up to f ive charges are show n inFigure 1.8. The mass of the protein remains the same, yet the m/z ratio varies depending upon the number ofcharges on the protein. Protein ionization is usually the result of protonation, w hich not only adds charge but alsoincreases the mass of the protein by the number of protons added. This effect on the m/z applies equally for anymechanism of molecular ionization resulting in a positively or negatively charged molecular ion, including theaddition or ejection of charge-carrying species other than protons (e.g. Na+ and Cs+). Multiple positive chargesare observed for proteins, w hile for oligonucleotides negative charging (w ith ESI) is typical.

Although electrospray mass spectrometers are equipped w ith softw are that w ill calculate molecular w eight,an understanding of how the computer makes such calculations from multiply-charged ions is beneficial.Equations 1.1 - 1.5 and Figure 1.9 offer a simple explana-tion, w here w e assume p1 and p2 are adjacent peaksand dif fer by a single charge, w hich is equivalent to the addition of a single proton.

Figure 1.8. A theoretical protein w ith a molecular w eight of 10,000 generates three different peaks w ith the ionscontaining 5, 4, and 3 charges, respectively. The mass spectrometer detects each of the protein ions at 2001,2501, and 3334, respectively.

Figure 1.9. The multiply charged ions of myoglobin generated from ESI. The different peaks represent dif ferentcharge states of myoglobin. The molecular w eight can be determined using Equations 1.1 - 1.3.

p = m/zp1 = (Mr + z1)/z1p2 = {Mr + (z1 - 1)}/(z1 - 1)

(1.1)(1.2)(1.3)

p is a peak in the mass spectrumm is the total mass of an ionz is the total chargeMr is the average mass of protein

p1 is the m/z value for p1p2 is the m/z value for p2z1 is the charge on peak p1

Equations 1.2 and 1.3 can be solved for the tw o unknow ns, Mr and z1.For the peaks in the mass spectrum of myoglobin show n in Figure 1.9, p1=1542, and p2=1696.

1542 z1 = Mr + z11696 (z1 - 1) = Mr + (z1 - 1)Solving the tw o equations: Mr = 16,951 Da for z1 = 11

(1.4)(1.5)

Electrospray Solvents

Many solvents can be used in ESI and are chosen based on the solubility of the compound of interest, thevolatility of the solvent and the solvent’s ability to donate a proton. Typically, protic primary solvents such asmethanol, 50/50 methanol/w ater, or 50/50 acetonitrile/H2O are used, w hile aprotic cosolvents, such as 10%DMSO in w ater, as w ell as isopropyl alcohol are used to improve solubility for some compounds. Although 100%w ater is used in ESI, w ater’s relatively low vapor pressure has a detrimental effect on sensitivity; better

sensitivity is obtained w hen a volatile organic solvent is added. Some compounds require the use of straightchloroform w ith 0.1% formic acid added to facilitate ionization. This approach, w hile less sensitive, can beeffective for otherw ise insoluble compounds.

Buffers such as Na+, K+, phosphate, and salts present a problem for ESI by low ering the vapor pressure ofthe droplets resulting in reduced signal through an increase in droplet surface tension resulting in a reduction ofvolatility (see Chapter 3 for quantitative information on salt effects). Consequently, volatile buffers such asammonium acetate can be used more effectively.

Table 1.3. Advantages and disadvantages of electrospray ionization (ESI)

Advantages Disadvantages

practical mass range of up to 70,000Dagood sensitivity w ith femtomole to lowpicomole sensitivity typicalsoftest ionization method, capable ofgenerating noncovalent complexes inthe gas phaseeasily adaptable to liquidchromatographyeasily adaptable to tandem massanalyzers such as ion traps and triplequadrupole instrumentsmultiple charging allow s for analysisof high mass ions w ith a relatively lowm/z range instrumentno matrix interference

the presence of salts and ion-pairingagents like TFA can reduce sensitivitycomplex mixtures can reducesensitivitysimultaneous mixture analysis can bepoormultiple charging can be confusingespecially in mixture analysissample purity is importantcarryover from sample to sample

Configuration of the Electrospray Ion Source

The off-axis ESI configuration now used in many instruments to introduce the ions into the analyzers (asshow n in Figure 1.10) has turned out to be very valuable for high flow rate applications. The primary advantageof this configuration is that the f low rates can be increased w ithout contaminating or clogging the inlet. Off-axisspraying is important because the entrance to the analyzer is no longer being saturated by solvent, thus keepingdroplets from entering and contaminating the inlet. Instead, only ions are directed tow ard the inlet. This makes ESIeven more compatible w ith LC/MS at the milliliter per minute f low rates.

Figure 1.10. An example of off-axis ESI.


Nanoelectrospray Ionization (NanoESI)

Low f low electrospray, originally described by Wilm and Mann, has been called nanoelectrospray, nanospray,and micro-electrospray. This ionization source is a variation on ESI, w here the spray needle has been made verysmall and is positioned close to the entrance to the mass analyzer (Figure 1.11). The end result of this rather

simple adjustment is increased eff iciency, w hich includes a reduction in the amount of sample needed.

Figure 1.11. Ion formation from electrospray ionization source. The electrospray ionization source uses a streamof air or nitrogen, heat, a vacuum, or a solvent sheath (often methanol) to facilitate desolvation of the droplets.Ejection of the ion occurs through a “Taylor cone” (central droplet) w here they are then electrostatically directedinto the mass analyzer.

The flow rates for nanoESI sources are on the order of tens to hundreds of nanoliters per minute. In order toobtain these low f low rates, nanoESI uses emitters of pulled and in some cases metallized glass or fused silicathat have a small orif ice (~5µ). The dissolved sample is added to the emitter and a pressure of ~30 PSI is appliedto the back of the emitter. Effusing the sample at very low f low rates allow s for high sensitivity. Also, theemitters are positioned very close to the entrance of the mass analyzer, therefore ion transmission to the massanalyzer is much more eff icient. For instance, the analysis of a 5 mM solution of a peptide by nanoESI w ould beperformed in 1 minute, consuming ~50 femtomoles of sample. The same experiment performed w ith normal ESI inthe same time period w ould require 5 picomoles, or 100 times more sample than for nanoESI. In addition, since thedroplets are typically smaller w ith nanoESI than normal ESI (Figure 1.11), the amount of evaporation necessaryto obtain ion formation is much less. As a consequence, nanoESI is more tolerant of salts and other impuritiesbecause less evaporation means the impurities are not concentrated dow n as much as they are in ESI.


Atmospheric Pressure Chemical Ionization

APCI has also become an important ionization source because it generates ions directly from solution and it iscapable of analyzing relatively nonpolar compounds. Similar to electrospray, the liquid eff luent of APCI (Figure1.12) is introduced directly into the ionization source. How ever, the similarity stops there. The droplets are notcharged and the APCI source contains a heated vaporizer, w hich facilitates rapid desolvation/vaporization of thedroplets. Vaporized sample molecules are carried through an ion-molecule reaction region at atmosphericpressure.

Figure 1.12. Atmospheric pressure chemical ionization (APCI) mass spectrometry.

APCI ionization originates from the solvent being excited/ionized from the corona discharge. Because thesolvent ions are present at atmospheric pressure conditions, chemical ionization of analyte molecules is veryefficient; at atmospheric pressure analyte molecules collide w ith the reagent ions frequently. Proton transfer (forprotonation MH+ reactions) occurs in the positive mode, and either electron transfer or proton loss, ([M-H]-) in thenegative mode. The moderating influence of the solvent clusters on the reagent ions, and of the high gaspressure, reduces fragmentation during ionization and results in primarily intact molecular ions. Multiple chargingis typically not observed presumably because the ionization process is more energetic than ESI. ( Back to the Table of Contents )

Atmospheric Pressure Photoionization

Atmospheric pressure photoionization (APPI) has recently become an important ionization source because itgenerates ions directly from solution w ith relatively low background and is capable of analyzing relativelynonpolar compounds. Similar to APCI, the liquid eff luent of APPI (Figure 1.13) is introduced directly into theionization source. The primary difference betw een APCI and APPI is that the APPI vaporized sample passesthrough ultra-violet light (a typical krypton light source emits at 10.0 eV and 10.6 eV). Often, APPI is much moresensitive than ESI or APCI and has been show n to have higher signal-to-noise ratios because of low erbackground ionization. Low er background signal is largely due to high ionization potential of standard solventssuch as methanol and w ater (IP 10.85 and 12.62 eV, respectively) w hich are not ionized by the krypton lamp.

Figure 1.13. Atmospheric pressure photoionization (APPI) mass spectrometry.

A disadvantage of both ESI and APCI is that they can generate background ions from solvents. Additionally,ESI is especially susceptible to ion suppression effects, and APCI requires vaporization temperatures rangingfrom 350-500° C, w hich can cause thermal degradation.

APPI induces ionization via tw o dif ferent mechanisms. The first is direct photoexcitation, allow ing for electronejection and the generation of the positive ion radical cation (M+). The APPI source imparts light energy that ishigher than the ionization potentials (IPs) of most target molecules, but low er than most of the IPs of air andsolvent molecules, thus removing them as interferants. In addition, because little excess energy is deposited inthe molecules, there is minimal fragmentation.

The second mechanism is atmospheric pressure photo-induced chemical ionization w hich is similar to APCI inthat it involves charge transfer to produce protonation (MH+) or proton loss ([M-H]-) to generate negative ions.

To initiate chemical ionization, a photoionizable reagent, also called a dopant, is added to the eluant. Uponphotoionization of the dopant, charge transfer occurs to the analyte. Typical dopants in positive mode includeacetone and toluene. Acetone also serves as a dopant in negative mode.

The ionization mechanism (M+ versus [M+H]+) that a molecule undergoes depends on the proton aff inity of theanalyte, the solvent, and the type of dopant used.


Matrix-Assisted Laser Desorption/Ionization

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) w as first introduced in 1988 byTanaka, Karas, and Hillenkamp. It has since become a w idespread analytical tool for peptides, proteins, and mostother biomolecules (oligonucleotides, carbohydrates, natural products, and lipids). The eff icient and directedenergy transfer during a matrix-assisted laser-induced desorption event provides high ion yields of the intactanalyte, and allow s for the measurement of compounds w ith sub-picomole sensitivity. In addition, the utility ofMALDI for the analysis of heterogeneous samples makes it very attractive for the mass analysis of complexbiological samples such as proteolytic digests.

Figure 1.14. The eff icient and directed energy transfer of the UV laser pulse during a MALDI event allow s forrelatively small quantities of sample (femtomole to picomole) to be analyzed. In addition, the utility of MALDI massspectrometry for the analysis of heterogeneous samples makes it very attractive for the mass analysis ofbiological samples.

While the exact desorption/ionization mechanism for MALDI is not know n, it is generally believed that MALDIcauses the ionization and transfer of a sample from the condensed phase to the gas phase via laser excitationand abalation of the sample matrix (Figure 1.14). In MALDI analysis, the analyte is f irst co-crystallized w ith alarge molar excess of a matrix compound, usually a UV-absorbing w eak organic acid. Irradiation of this analyte-matrix mixture by a laser results in the vaporization of the matrix, w hich carries the analyte w ith it. The matrixplays a key role in this technique. The co-crystallized sample molecules also vaporize, but w ithout having todirectly absorb energy from the laser. Molecules sensitive to the laser light are therefore protected from directUV laser excitation.

MALDI matrix -- A nonvolatile solid material facilitates the desorption and ionization processby absorbing the laser radiation. As a result, both the matrix and any sample embedded in thematrix are vaporized. The matrix also serves to minimize sample damage from laser radiationby absorbing most of the incident energy.

Once in the gas phase, the desorbed charged molecules are then directed electrostatically from the MALDIionization source into the mass analyzer. Time-of-f light (TOF) mass analyzers are often used to separate theions according to their mass-to-charge ratio (m/z). The pulsed nature of MALDI is directly applicable to TOFanalyzers since the ion’s initial time-of-f light can be started w ith each pulse of the laser and completed w hen theion reaches the detector.

Several theories have been developed to explain desorption by MALDI. The thermal-spike model proposes thatthe ejection of intact molecules is attributed to poor vibrational coupling betw een the matrix and analyte, w hichminimizes vibrational energy transfer from the matrix to the vibrational modes of the analyte molecule, therebyminimizing fragmentation. The pressure pulse theory proposes that a pressure gradient from the matrix is creatednormal to the surface and desorption of large molecules is enhanced by momentum transfer from collisions w iththese fast moving matrix molecules. It is generally thought that ionization occurs through proton transfer orcationization during the desorption process.

The utility of MALDI for biomolecule analyses lies in its ability to provide molecular w eight information on intactmolecules. The ability to generate accurate information can be extremely useful for protein identif ication andcharacterization. For example, a protein can often be unambiguously identif ied by the accurate mass analysis ofits constituent peptides (produced by either chemical or enzymatic treatment of the sample).

Table 1.4. Advantages and disadvantages of Matrix-Assisted Laser Desorption/Ionization (MALDI).

Advantages Disadvantages

practical mass range of up to 300,000Da. Species of much greater masshave been observed using a highcurrent detector;typical sensitivity on the order of lowfemtomole to low picomole. Attomolesensitivity is possible;soft ionization w ith little to nofragmentation observed;tolerance of salts in millimolarconcentrations;suitable for the analysis of complexmixtures.

matrix background, w hich can be aproblem for compounds below a massof 700 Da. This backgroundinterferences is highly dependent onthe matrix material;possibility of photo-degradation bylaser desorption/ionization;acidic matrix used in MALDI my causedegradation on some compounds.

Figure 1.15. Commonly used MALDI matrices and a MALDI plate show ing the matrix deposition. One of theadvantages of MALDI is that multiple samples can be prepared at the same time, as seen w ith this multisampleplate.

Sample-matrix preparation procedures greatly influence the quality of MALDI mass spectra ofpeptides/proteins (Figure 1.15). Among the variety of reported preparation methods, the dried-droplet method isthe most frequently used. In this case, a saturated matrix solution is mixed w ith the analyte solution, giving amatrix-to-sample ratio of about 5000:1. An aliquot (0.5-2.0 µL) of this mixture is then applied to the sample targetw here it is allow ed to dry. Below is an example of how the dried-droplet method is performed:

Pipet 0.5 µL of sample to the sample plate.Pipet 0.5 µL of matrix to the sample plate.Mix the sample and matrix by draw ing the combined droplet in and out of the pipette.Allow to air dry.

For peptides, small proteins and most compounds: A saturated solution of α-cyano-4-hydroxycinnamic acid in 50:50 ACN:H2O w ith 0.1% TFA.For proteins and other large molecules: a saturated solution of sinapinic acid in 50:50 ACN:H2Ow ith 0.1% TFA.For glycopeptides/proteins and small compounds: a saturated solution of 2,5-dihydroxy benzoicacid (DHB) in 50:50 ACN:H2O.

Alternatively, samples can be prepared in a stepw ise manner. In the thin layer method, a homogeneous matrix“f ilm” is formed on the target f irst, and the sample is then applied and absorbed by the matrix. This method yieldsgood sensitivity, resolving pow er, and mass accuracy. Similarly, in the thick-layer method, nitrocellulose (NC) is

used as the matrix additive; once a uniform NC-matrix layer is obtained on the target, the sample is applied. Thispreparation method suppresses alkali adduct formation and signif icantly increases the detection sensitivity,especially for peptides and proteins extracted from gels. The sandw ich method is another variant in thiscategory. A thin layer of matrix crystals is prepared as in the thin-layer method, follow ed by the subsequentaddition of droplets of (a) aqueous 0.1% TFA, (b) sample and (c) matrix.


Desorption/Ionization on Silicon (DIOS)

DIOS is a matrix-free method that uses pulsed laser desorption/ionization on silicon (Figure 1.16). Structuredsilicon surfaces such as porous silicon or silicon nanow ires are UV-absorbing semiconductors w ith a largesurface area (hundreds of m2/cm3). For its application to laser desorption/ionization mass spectrometry, thestructure of structured silicon provides a scaffold for retaining solvent and analyte molecules, and the UVabsorptivity affords a mechanism for the transfer of the laser energy to the analyte. This fortuitous combinationof characteristics allow s DIOS to be useful for a large variety of biomolecules including peptides, carbohydrates,and small organic compounds of various types. Unlike other direct, matrix-free desorption techniques, DIOSenables desorption/ionization w ith little or no analyte degradation.

DIOS has a great deal in common w ith MALDI. Instrumentation and acquisition using DIOS-MS requires onlyminor adjustments to the MALDI setup; the chips are simply aff ixed to a machined MALDI plate and inserted intothe spectrometer. The same w avelength of laser light (337 nm) typically employed in MALDI is effective for DIOS.While DIOS is comparable to MALDI w ith respect to its sensitivity, it has several advantages due to the lack ofinterfering matrix: low background in the low mass range; uniform deposition of aqueous samples; and simplif iedsample handling. In addition, the chip-based format can be adapted to automated sample handling, w here thelaser rapidly scans from spot to spot. DIOS could thus accelerate and simplify high-throughput analysis of lowmolecular w eight compounds, as MALDI has done for macromolecules. Because the masses of many lowmolecular w eight compounds can be measured, DIOS-MS can be applied to the analysis of small moleculetransformations, both enzymatic and chemical.

In a number of recent advances w ith DIOS-MS, the modification of the silicon surface w ith f luorinated silyatingreagents have allow ed for ultra-high sensitivity in the yoctomole range (Figure 1.16).

Figure 1.16. Desorption/Ionization on Silicon (DIOS) uses UV laser pulse from a structured silicon surface togenerate intact gas phase ions. DIOS allow s for small quantities of sample to be analyzed, 800 yoctomoles (480molecules) of des-arg-bradykinin has been detected. In addition, DIOS mass spectrometry is useful for theanalysis of heterogeneous samples and small molecules. On the left is a picture of a DIOS chip; the dark spotsrepresent porous silicon. On the right is a diagrammatic representation of the DIOS event from a chip.


Fast Atom/Ion Bombardment

Fast atom ion bombardment, or FAB, is an ionization source similar to MALDI in that it uses a matrix and ahighly energetic beam of particles to desorb ions from a surface. It is important, how ever, to point out thedifferences betw een MALDI and FAB. For MALDI, the energy beam is pulsed laser light, w hile FAB uses a

continuous ion beam. With MALDI, the matrix is typically a solid crystalline, w hereas FAB typically has a liquidmatrix. It is also important to note that FAB is about 1000 times less sensitive than MALDI.

Figure 1.17. Fast atom bombardment (FAB) mass spectrometry, also know n as liquid secondary ion massspectrometry (LSIMS).

Fast atom bombardment is a soft ionization source w hich requires the use of a direct insertion probe forsample introduction, and a beam of Xe neutral atoms or Cs+ ions to sputter the sample and matrix from the directinsertion probe surface. It is common to detect matrix ions in the FAB spectrum as w ell as the protonated orcationized (i.e. M + Na+) molecular ion of the analyte of interest.

FAB matrix -- Facilitating the desorption and ionization process, the FAB matrix is a nonvolatileliquid material that serves to constantly replenish the surface w ith new sample as it isbombarded by the incident ion beam. By absorbing most of the incident energy, the matrix alsominimizes sample degradation from the high-energy particle beam.

Tw o of the most common matrices used w ith FAB are m-nitrobenzyl alcohol and glycerol.

m-nitrobenzyl alcohol (NBA) glycerol

The fast atoms or ions impinge on or collide w ith the matrix causing the matrix and analyte to be desorbed intothe gas phase. The sample may already be charged and subsequently transferred into the gas phase by FAB, orit may become charged during FAB desorption through reactions w ith surrounding molecules or ions. Once in thegas phase, the charged molecules can be propelled electrostatically to the mass analyzer.


Electron Ionization

Electron ionization is one of the most important ionization sources for the routine analysis of small,hydrophobic, thermally stable molecules and is still w idely used. Because EI usually generates numerousfragment ions it is a “hard” ionization source. How ever, the fragmentation information can also be very useful.For example, by employing databases containing over 200,000 electron ionization mass spectra, it is possible toidentify an unknow n compound in seconds (provided it exists in the database). These databases, combined w ithcurrent computer storage capacity and searching algorithms, allow for rapid comparison w ith these databases(such as the NIST database), thus greatly facilitating the identif ication of small molecules.

Figure 1.18. Electron ionization (EI) mass spectrometry.

The electron ionization source is straightforw ard in design (Figure 1.18). The sample must be delivered as agas w hich is accomplished by either “boiling off” the sample from a probe via thermal desorption, or byintroduction of a gas through a capillary. The capillary is often the output of a capillary column from gaschromatography instrumentation. In this case, the capillary column provides separation (this is also know n as gaschromatography mass spectrometry or GC/MS). Desorption of both solid and liquid samples is facilitated by heatas w ell as the vacuum of the mass spectrometer. Once in the gas phase the compound passes into an electronionization source, w here electrons excite the molecule, thus causing electron ejection ionization andfragmentation.

The utility of electron ionization decreases signif icantly for compounds above a molecular w eight of 400 Dabecause the required thermal desorption of the sample often leads to thermal decomposition before vaporization

because the required thermal desorption of the sample often leads to thermal decomposition before vaporizationis able to occur. The principal problems associated w ith thermal desorption in electron ionization are 1) involatilityof large molecules, 2) thermal decomposition, and 3) excessive fragmentation.

The method, or mechanism, of electron ejection for positive ion formation proceeds as follow s:

The sample is thermally vaporized.

Electrons ejected from a heated filament are accelerated through an electric f ield at 70 V to form acontinuous electron beam.

The sample molecule is passed through the electron beam.

The electrons, containing 70 V of kinetic energy (70 electron volts or 70 eV), transfer some of theirkinetic energy to the molecule. This transfer results in ionization (electron ejection) w ith the ion internallyretaining usually no more than 6 eV excess energy.M + e- (70 eV) → M+ (~5 eV) + 2e- (~65 eV)

Excess internal energy (6 eV) in the molecule leads to some degree of fragmentation.M+ → molecular ions + fragment ions + neutral fragments

Electron capture is usually much less eff icient than electron ejection, yet it is sometimes used in the follow ingw ay for high sensitivity w ork w ith compounds having a high electron aff inity: M + e- → M-.


Chemical Ionization

Chemical Ionization (CI) is applied to samples similar to those analyzed by EI and is primarily used to enhancethe abundance of the molecular ion. Chemical ionization uses gas phase ion-molecule reactions w ithin thevacuum of the mass spectrometer to produce ions from the sample molecule. The chemical ionization process isinitiated w ith a reagent gas such as methane, isobutane, or ammonia, w hich is ionized by electron impact. Highgas pressure in the ionization source results in ion-molecule reactions betw een the reagent gas ions and reagentgas neutrals. Some of the products of the ion-molecule reactions can react w ith the analyte molecules toproduce ions.

A possible mechanism for ionization in CI occurs as follow s:

Reagent (R) + e- → R+ + 2 e-

R+ + RH → RH+ + R

RH+ + Analyte (A) → AH+ + R

In contrast to EI, an analyte is more likely to provide a molecular ion w ith reduced fragmentation using CI.How ever, similar to EI, samples must be thermally stable since vaporization w ithin the CI source occurs throughheating.

Negative chemical ionization (NCI) typically requires an analyte that contains electron-capturing moieties (e.g.,f luorine atoms or nitrobenzyl groups). Such moieties signif icantly increase the sensitivity of NICI, in some cases100 to 1000 times greater than that of electron ionization (EI). NCI is probably one of the most sensitivetechniques and is used for a w ide variety of small molecules w ith the caveat that the molecules are oftenchemically modified w ith an electron-capturing moiety prior to analysis.

Figure 1.19. The pentafluorobenzyl trimethyl silyl ether derivatives of steroids make them more amenable to highsensitivity measurements using negative chemical ionization.

While most compounds w ill not produce negative ions using EI or CI, many important compounds can producenegative ions and, in some cases, negative EI or CI mass spectrometry is more sensitive and selective thanpositive ion analysis. In fact, compounds like steroids are modified (Figure 1.19) to enhance NCI.

As mentioned, negative ions can be produced by electron capture, and in negative chemical ionization abuffer gas (such as methane) can slow dow n the electrons in the electron beam allow ing them to be capturedby the analyte molecules. The buffer gas also stabilizes the excited anions and reduces fragmentation.Therefore, NCI is in actuality an electron capture process and not w hat w ould traditionally be defined as a“chemical ionization” process.

Table 1.5. General Comparison of Ionization Sources.

IonizationSource

TypicalMassRange

(Da)

MatrixInterference

Degradation ComplexMixtures

LC/MSAmenable

Sensitivity

ElectrosprayIonization(ESI)

70,000 none none somew hatlimited

excellent highfemtomoleto lowpicomole

Comments Excellent LC/MS tool; low salt tolerance (low millimolar); multiple charging useful, but signif icantsuppression w ith mixture occurs; low tolerance of mixtures; soft ionization (little fragmentationobserved).

NanoESI 70,000 none none somew hatlimited butbetter thanESI

OK but low f lowrates canpresent aproblem

highzeptomoleto lowfemtomole

Comments Very sensitive and very low f low rates; applicable to LC/MS; but low flow rates requirespecialized systems; has reasonable salt tolerance (low millimolar); multiple charging useful butsignif icant suppression can occur w ith mixtures; reasonable tolerance of mixtures; softionization (little fragmentation observed).

APCI 1,200 none thermaldegradation

somew hatamenable

excellent highfemtomole

Comments Excellent LC/MS tool; low salt tolerance (low millimolar); useful for hydrophobic materials.

APPI 1,200 none photodissociation

amenable excellent highfemtomole

Comments Excellent LC/MS tool; low salt tolerance (low millimolar); useful for hydrophobic materials.

MALDI 300,000 yes photodegradation andmatrix reactions

good forcomplexmixtures

possible low to highfemtomole

Comments Somew hat tolerant of salts; excellent sensitivity; matrix background can be a problem for lowmass ions; soft ionization (little fragmentation observed); photo degradation possible; suitablefor complex mixtures. Limited multiple charging occurs so MS/MS data is not extensive.

DIOS 3,000 none photodegradation

good forcomplexmixtures

possible lowfemtomoleto highyoctomole

Comments Somew hat tolerant of salts; excellent sensitivity; soft ionization (little fragmentation observed);photo degradation possible; suitable for complex mixtures and small molecules.

FAB 7,000 yes matrix reactionsand somethermaldegradation

somew hatamenable

very limited nanomole

Comments Relatively insensitive; little fragmentation; soft ionization; high salt tolerance to 0.01M solubilityw ith matrix required.

ElectronIonization(EI)

500 none thermaldegradation

limitedunless usedw ith GC/MS

very limited picomole

Comments Good sensitivity; unique fragmentation data generated; National Institute of Science andTechnology (NIST) database (>100,000 compounds) available to compare fragmentation data;thermal decomposition a major problem for biomolecules; limited mass range due to thermaldesorption requirement.

ChemicalIonization(CI)

500 none thermaldegradation

limitedunless usedw ith GC/MS

very limited picomole

Comments Offers a softer ionization approach over EI yet still requires thermal desorption; negative CIparticularly sensitive for perf lourinated derivatives; a limited but pow erful approach for certainderivatized molecules such as steroids.

Summary

The mass spectrometer as a w hole can be separated into distinct sections that include the sample inlet, ionsource, mass analyzer, and detector. A sample is introduced into the mass spectrometer and is then ionized. Theion source produces ions either by electron ejection, electron capture, cationization, deprotonation or the transferof a charged molecule from the condensed to the gas phase. MALDI and ESI have had a profound effect onmass spectrometry because they generate charged intact biomolecules into the gas phase. In comparison toother ionization sources such as APCI, EI, FAB, and CI, the techniques of MALDI and ESI have greatly extendedthe analysis capabilities of mass spectrometry to a w ide range of compounds w ith detection capabilities rangingfrom the picomole to the zeptomole level.

Useful References

Dole M, Mack LL, Hines RL, Mobley RC, Ferguson LD, Alice MB. Molecular beams of macroions. Journal ofChemical Physics. 1968, 49:5, 2240.

Whitehouse CM, Dreyer RN, Yanashita M, Fenn JB. Electrospray interface for liquid chromatographs and massspectrometers. Anal. Chem. 1985, 57, 675-679.

Tanaka K, Waki H, Ido Y, Akita S, Yoshida Y, Yoshida T. Protein and polymer analysis up to m/z 100,000 bylaser ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 1988, 2, 151.

Karas M & Hillenkamp F. Laser desorption ionization of proteins with molecular mass exceeding 10,000 Daltons.Anal. Chem. 1988, 60, 2299.

Bruins AP. Mechanistic aspects of electrospray ionization. J. Chromatogr. A, 1998, 795, 345-357.

Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM. Electrospray ionization - principles and practice. MassSpectrometry Review s. 1990, 9, 37.

McLafferty FW & Turecek F. Interpretation of Mass Spectra. 4th ed. Mill Valley, Calif. : University Science Books,1993.

Cole R (Editor). Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation, and Applications.New York: Wiley and Sons, 1997.

Cole RB. Some tenets pertaining to electrospray ionization mass spectrometry. J. Mass Spectrom. 2000, 35,763-772.

Kebarle P. A brief overview of the present status of the mechanisms involved in electrospray massspectrometry. J. Mass Spectrom. 2000, 35, 804-817.

Gaskell SJ. Electrospray: principles and practice. J. Mass Spectrom. 2000, 35, 677-688.

Cech NB and Enke CG. Practical implications of some recent studies in electrospray ionization fundamentals.Mass Spectrom. Rev. 2001, 20, 362-387.


Mass Analyzers

With the advent of ionization sources that can vaporize and ionize biomolecules, it has become necessary toimprove mass analyzer performance w ith respect to speed, accuracy, and resolution (Figure 2.1). Morespecif ically, quadrupoles, quadrupole ion traps, time-of-f light (TOF), time-of-f light ref lectron, and ion cyclotronresonance (ICR) mass analyzers have undergone numerous modifications/improvements over the past decade inorder to be interfaced w ith MALDI and ESI. The biggest challenge came in the ionization of interfacingatmospheric pressure sources (760 torr) to analyzers maintained at 10-6 to 10-11 torr, a remarkable pressuredifferential of more than 9 orders of magnitude. This chapter w ill focus on the principles of operation and currentperformance capabilities of mass analyzers, w hile brief ly touching on ion detectors and the concept of vacuumin a mass spectrometer.

Mass Analysis

Analytical instruments in general have variations in their capabilities as a result of their individual design andintended purpose. This is also true for mass spectrometers. While all mass spectrometers rely on a massanalyzer, not all analyzers operate in the same w ay; some separate ions in space w hile others separate ions bytime. In the most general terms, a mass analyzer measures gas phase ions w ith respect to their mass-to-chargeratio (m/z), w here the charge is produced by the addition or loss of a proton(s), cation(s), anion(s) orelectron(s). The addition of charge allow s the molecule to be affected by electric f ields thus allow ing its massmeasurement. This is an important aspect to remember about mass analyzers -- they measure the m/z ratio, notthe mass. It is often a point of confusion because if an ion has multiple charges, the m/z w ill be signif icantly lessthan the actual mass (Figures 1.8 and 1.9). For example, a doubly charged peptide ion of mass 976.5 Daltons(Da) (C37H68N16O14

2+) has an m/z of 488.3.

Figure 2.1. The effect of resolution upon mass accuracy. The overlaid spectra w ere calculated for the samemolecular formula (C101H145N34O44) at resolutions of 200, 2500, and infinity (∞).

Multiple charging is especially common w ith electrospray ionization, yielding numerous peaks that correspondto the same species yet are observed at different m/z.

The first mass analyzers, made in the early 1900’s, used magnetic f ields to separate ions according to theirradius of curvature through the magnetic f ield. The design of modern analyzers has changed signif icantly in thelast f ive years, now offering much higher accuracy, increased sensitivity, broader mass range, and the ability togive structural information. Because ionization techniques have evolved, mass analyzers have been forced tochange in order to meet the demands of analyzing a w ide range of biomolecular ions w ith part per million massaccuracy and sub femtomole sensitivity. The characteristics (Table 2.1) of these mass analyzers w ill becovered in this chapter.

Table 2.1Mass Analyzers EventQuadrupole scan radio frequency fieldQuadrupole Ion Trap scan radio frequency fieldTime-of-Flight (TOF) time-of-f light correlated directly to ion's m/zTime-of-Flight Reflectron time-of-f light correlated directly to ion's m/zQuad-TOF radio frequency field scanning and time-of-f lightMagnetic Sector magnetic f ield affects radius of curvature of ionsFourier Transform Ion Cyclotron Resonance MS translates ion cyclotron motion to m/z (FTMS)

Performance Characteristics

The performance of a mass analyzer can typically be defined by the follow ing characteristics: accuracy,resolution, mass range, tandem analysis capabilities, and scan speed.

Accuracy

This is the ability w ith w hich the analyzer can accurately provide m/z information and is largely a function ofan instrument’s stability and resolution. For example, an instrument w ith 0.01% accuracy can provide informationon a 1000 Da peptide to ±0.1 Da or a 10,000 Da protein to ±1.0 Da. The accuracy varies dramatically fromanalyzer to analyzer depending on the analyzer type and resolution. An alternative means of describingaccuracy is using part per million (ppm) terminology, w here 1000 Da peptide to ±0.1 Da could also be describedas 1000.00 Da peptide to ± 100 ppm.

Resolution (Resolving Power)

Resolution is the ability of a mass spectrometer to distinguish betw een ions of dif ferent mass-to-chargeratios. Therefore, greater resolution corresponds directly to the increased ability to differentiate ions. The mostcommon definition of resolution is given by the follow ing equation:

Resolution = M/ΔM Equation 2.1

w here M corresponds to m/z and ΔM represents the full w idth at half maximum (FWHM). An example ofresolution measurement is show n in Figure 2.2 w here the peak has an m/z of 500 and a FWHM of 1. Theresulting resolution is M/ΔM = 500/1 = 500.

Figure 2.2. The resolution is determined by the measurement of peak’s m/z and FWHM , in this case m/z = 500and the FWHM = 1.

The analyzer’s resolving pow er does, to some extent, determine the accuracy of a particular instrument, ascharacterized in Figure 2.2. The average mass of a molecule is calculated using the w eighted average mass ofall isotopes of each constituent element of the molecule. The monoisotopic mass is calculated using the mass ofthe elemental isotope having the greatest abundance for each constituent element. If an instrument cannotresolve the isotopes it w ill generate a broad peak w ith the center representing the average mass. Higherresolution can offer the benefits of separating an ion’s individual isotopes or the narrow ing of peaks allow s amore accurate determination of its position.

Mass Range

This is the m/z range of the mass analyzer. For instance, quadrupole analyzers typically scan up to m/z 3000.A magnetic sector analyzer typically scans up to m/z 10,000 and time-of-f light analyzers have virtually unlimitedm/z range.

Tandem Mass Analysis (MS/MS or MSn)

This is the ability of the analyzer to separate different molecular ions, generate fragment ions from a selectedion, and then mass measure the fragmented ions. The fragmented ions are used for structural determination oforiginal molecular ions.

Typically, tandem MS experiments are performed by colliding a selected ion w ith inert gas molecules such asargon or helium, and the resulting fragments are mass analyzed. Tandem mass analysis is used to sequencepeptides, and structurally characterize carbohydrates, small oligo-nucleotides, and lipids.

Scheme 2.1. Tandem mass spectrometry analysis.

The term “tandem” mass analysis comes from the events being either tandem in space or tandem in time.Tandem mass analysis in space is performed by consecutive analyzers w hereas tandem mass analysis in timeis performed w ith the same analyzer, w hich isolates the ion of interest, fragments it, and analyzes the fragment

ions. Tandem analysis characteristics are summarized for the dif ferent analyzers in Table 2.2.

Scan Speed

This refers to the rate at w hich the analyzer scans over a particular mass range. Most instruments requireseconds to perform a full scan, how ever this can vary w idely depending on the analyzer. Time-of-f lightanalyzers, for example, complete analyses in milliseconds or less.

Mass Analyzers

It is clear from Chapter 1 that ESI and MALDI are quite different in terms of how ions are generated. ESIcreates ions in a continuous stream from charged droplets under atmospheric pressure conditions and ions arecreated in a continuous stream, for these reasons quadrupoles presented an w ell-suited analyzer for ESI sincethey are both tolerant of relatively high pressures (~10-5 torr) and they are capable of continuously scanning theESI ion stream. MALDI, on the other hand, generates ions from short, nanosecond laser pulses and is readilycompatible w ith time-of-f light mass analysis, w hich measures precisely timed ion packets such as thosegenerated from a laser pulse. The most common analyzers are discussed in this section w ith a description oftheir respective advantages and disadvantages.

Quadrupoles

Quadrupole mass analyzers (Figure 2.3) have been used w ith EI sources since the 1950’s and are still themost common mass analyzers in existence today. Interestingly, quadrupole mass analyzers have found newutility in their capacity to interface w ith ESI and APCI. Quadrupoles offer three main advantages. They toleraterelatively high pressures. Secondly, quadrupoles have a signif icant mass range w ith the capability of analyzingup to an m/z of 4000, w hich is useful because electrospray ionization of proteins and other biomoleculescommonly produce charge distributions from m/z 1000 to 3500. Finally, quadrupole mass spectrometers arerelatively low cost instruments. Considering the mutually complementary features of ESI and quadrupoles, it is notsurprising that the f irst successful commercial electrospray instruments w ere coupled w ith quadrupole massanalyzers.

Figure 2.3. Schematic diagram show ing arrangement of quadrupole rods and electrical connection to RFgenerator; a DC potential (not show n) is also superimposed on the rods. A cross-section of a quadrupole massanalyzer taken as it analyzes for m/z 100, 10, and 1000, respectively. It is important to note that both the DC andRF fields are the same in all three cases and only ions w ith m/z = 100 (top example) traverse the total length ofthe quadrupole and reach the detector; the other ions are f iltered out.

Quadrupole mass analyzers are connected in parallel to a radio frequency (RF) generator and a DC potential.At a specif ic RF field, only ions of a specif ic m/z can pass through the quadrupoles as show n in Figure 2.3,w here only the ion of m/z 100 is detected. In all three cases in Figure 2.3 the DC and RF fields are the same.Therefore by scanning the RF field a broad m/z range (typically 100 to 4000) can be achieved in approximatelyone second

one second.

In order to perform tandem mass analysis w ith a quadrupole instrument, it is necessary to place threequadrupoles in series. Each quadrupole has a separate function: the f irst quadrupole (Q1) is used to scanacross a preset m/z range and select an ion of interest. The second quadrupole (Q2), also know n as thecollision cell, focuses and transmits the ions w hile introducing a collision gas (argon or helium) into the f light pathof the selected ion. The third quadrupole (Q3) serves to analyze the fragment ions generated in the collision cell(Q2) (Figure 2.4). A stepw ise example of collision-induced dissociation (CID), is show n in Scheme 2.1.

Figure 2.4. A triple quadrupole ESI mass spectrometer possesses ion selection and fragmentation capabilitiesallow ing for tandem mass spectra.


Quadrupole Ion Trap

The ion trap mass analyzer show n in Figure 2.5 (roughly the size of a tennis ball) w as conceived at thesame time as the quadrupole mass analyzer by the same person, Wolfgang Paul. Incidentally, the physics behindboth of these analyzers is similar. How ever, in an ion trap, rather than passing through a quadrupole analyzerw ith a superimposed radio frequency field, the ions are trapped in a radio frequency quadrupole f ield. Onemethod of using an ion trap for mass spectrometry involves generating ions internally w ith EI, follow ed by massanalysis. Another, more popular, method of using an ion trap for mass spectrometry involves generating ionsexternally w ith ESI or MALDI and using ion optics for sample injection into the trapping volume. The quadrupole iontrap typically consists of a ring electrode and tw o hyperbolic endcap electrodes (Figure 2.5). The motion of theions induced by the electric f ield on these electrodes allow s ions to be trapped or ejected from the ion trap. In thenormal mode, the radio frequency is scanned to resonantly excite and therefore eject ions through small holes inthe endcap to a detector. As the RF is scanned to higher frequencies, higher m/z ions are excited, ejected, anddetected.

A very useful feature of ion traps is that it is possible to isolate one ion species by ejecting all others from thetrap. The isolated ions can subsequently be fragmented by collisional activation and the fragments detected. Theprimary advantage of quadrupole ion traps is that multiple collision induced dissociation experiments can beperformed quickly w ithout having multiple analyzers, such that real time LC-MS/MS is now routine. Otherimportant advantages of quadrupole ion traps include their compact size, and their ability to trap and accumulateions to provide a better ion signal.

Quadrupole ion traps have been utilized in a number of applications ranging from electrospray ionization MSn(Figure 2.5) of biomolecules to their more recent interface w ith MALDI. MSn allow s for multiple MS/MSexperiments to be performed on subsequent fragment ions, providing additional fragmentation information. Yet,ion traps most important application has been in the characterization of proteins. LC-MS/MS experiments areperformed on proteolytic digests w hich provide both MS and MS/MS information. This information allow s forprotein identif ication and post-translational modification characterization. The mass range (~4000 m/z) ofcommercial LC-traps is w ell matched to m/z values generated from the electrospray ionization of peptides andthe resolution allow s for charge state identif ication of multiply-charged peptide ions. Quadrupole ion trap massspectrometers can analyze peptides from a tryptic digest present at the 20-100 fmol level. Another asset of theion trap technique for peptide analysis is the ability to perform multiple stages of mass spectrometry, w hich cansignif icantly increase the amount of structural information.

Figure 2.5. Ions inside a 3D ion trap mass analyzer can be analyzed to produce a mass spectrum, or a particularion can be trapped inside and made to undergo collisions to produce fragmentation information.


Linear Ion Trap

The linear ion trap differs from the 3D ion trap (Figure 2.6) as it confines ions along the axis of a quadrupolemass analyzer using a tw o-dimensional (2D) radio frequency (RF) f ield w ith potentials applied to end electrodes.

The primary advantage to the linear trap over the 3D trap is the larger analyzer volume lends itself to a greaterdynamic ranges and an improved range of quantitative analysis.

Figure 2.6. A linear ion trap mass analyzer confines the ions along the axis of quadrupoles using a 2D radiofrequency and stopping potentials on the end electrodes.

Ion Trap’s Limitations: Precursor ion scanning, “1/3 rule” & Dynamic range

Given the pow er of the ion trap the major limitations of this device that keep it from being the ultimate tool forpharmacokinetics and proteomics include the follow ing: 1) the ability to perform high sensitivity triple quadrupole-type precursor ion scanning and neutral loss scanning experiments is not possible w ith ion traps. 2) The upperlimit on the ratio betw een precursor m/z and the low est trapped fragment ion is ~0.3 (also know n as the “onethird rule”). An example of the one third rule is that fragment ions of m/z 900 w ill not be detected below m/z 300,presenting a signif icant limitation for de novo sequencing of peptides. 3) The dynamic range of ion traps arelimited because w hen too many ions are in the trap, space charge effects diminish the performance of the iontrap analyzer. To get around this, automated scans can rapidly count ions before they go into the trap, thereforelimiting the number of ions getting in. Yet this approach presents a problem w hen an ion of interest isaccompanied by a large background ion population.


Double-Focusing Magnetic Sector

The earliest mass analyzers separated ions w ith a magnetic f ield. In magnetic analysis, the ions areaccelerated into a magnetic f ield using an electric f ield. A charged particle traveling through a magnetic f ield w illtravel in a circular motion w ith a radius that depends on the speed of the ion, the magnetic f ield strength, and theion’s m/z. A mass spectrum is obtained by scanning the magnetic f ield and monitoring ions as they strike a f ixedpoint detector. A limitation of magnetic analyzers is their relatively low resolution. In order to improve this,magnetic instruments w ere modified w ith the addition of an electrostatic analyzer to focus the ions. These arecalled double-sector or tw o-sector instruments. The electric sector serves as a kinetic energy focusing elementallow ing only ions of a particular kinetic energy to pass through its f ield irrespective of their mass-to-charge ratio.Thus, the addition of an electric sector allow s only ions of uniform kinetic energy to reach the detector, therebydecreasing the kinetic energy spread, w hich in turn increases resolution. It should be noted that thecorresponding increase in resolution does have its costs in terms of sensitivity. These double-focusing (Figure2.7) mass analyzers are used w ith ESI, FAB and EI ionization, how ever they are not w idely used today primarilydue to their large size and the success of time-of-f light, quadrupole and FTMS analyzers w ith ESI and MALDI.

Figure 2.7. A tw o-sector double-focusing instrument.


Quadrupole Time-of-Flight Tandem MS

The linear time-of-f light (TOF) mass analyzer (Figure 2.7) is the simplest mass analyzer. It has enjoyed arenaissance w ith the invention of MALDI and its recent application to electrospray and even gas chromatographyelectron ionization mass spectrometry (GC/MS). Time-of-f light analysis is based on accelerating a group of ionsto a detector w here all of the ions are given the same amount of energy through an accelerating potential.Because the ions have the same energy, but a different mass, the lighter ions reach the detector f irst because oftheir greater velocity, w hile the heavier ions take longer due to their heavier masses and low er velocity. Hence,the analyzer is called time-of-f light because the mass is determined from the ions’ time of arrival. Mass, charge,and kinetic energy of the ion all play a part in the arrival time at the detector. Since the kinetic energy (KE) of theion is equal to 1/2 mv2, the ion’s velocity can be represented as v = d/t = (2KE/m)1/2. The ions w ill travel a givendistance d, w ithin a time t, w here t is dependent upon the mass-to-charge ratio (m/z). In this equation, v = d/t =(2KE/m)1/2, assuming that z = 1. Another representation of this equation to more clearly present how mass isdetermined is m = 2t2 KE/d2 w here KE is constant.

Figure 2.8. Time-of-f light and time-of-f light reflectron mass analyzers. The TOF analyzer has virtually unlimitedmass range, w hile the TOF ref lectron has mass range up to m/z ~10,000. It should be noted that most detectors

have a limited mass range.

The time-of-f light (TOF) reflectron (Figure 2.8) is now w idely used for ESI, MALDI, and more recently forelectron ionization in GC/MS applications. It combines time-of-f light technology w ith an electrostatic mirror. Thereflectron serves to increase the amount of time (t) ions need to reach the detector w hile reducing their kineticenergy distribution, thereby reducing the temporal distribution Δt. Since resolution is defined by the mass of apeak divided by the w idth of a peak or m/Δm (or t/Δt since m is related to t), increasing t and decreasing Δtresults in higher resolution. Therefore, the TOF reflectron offers high resolution over a simple TOF instrument byincreasing the path length and kinetic energy focusing through the ref lectron. It should be noted that theincreased resolution (typically above 5000) and sensitivity on a TOF reflectron does decrease signif icantly athigher masses (typically above 5000 m/z).

Another type of tandem mass analysis, MS/MS, is also possible w ith MALDI TOF reflectron mass analyzers.MS/MS is accomplished by taking advantage of MALDI fragmentation that occurs follow ing ionization, or post-source decay (PSD). Time-of-f light instruments alone w ill not separate post-ionization fragment ions from thesame precursor ion because both the precursor and fragment ions have the same velocity and thus reach thedetector at the same time. The reflectron takes advantage of the fact that the fragment ions have different kineticenergies and separates them based on how deeply the ions penetrate the reflectron field, thus producing afragment ion spectrum (Figure 2.9 and 2.10).

Figure 2.9. A MALDI time-of-f light ref lectron mass analyzer and its ability to improve resolution over time-of-f lightanalysis w ith the reflectron. The TOF reflectron mass analyzer w ith an ESI ion source has also gained w ide usedue to the fast acquisition rates (milliseconds), good mass range (up to ~10,000 m/z) and accuracy on the orderof 5 part per million (ppm).

It should be noted that electrospray has also been adapted to TOF reflectron analyzers, w here the ions fromthe continuous ESI source can be stored in the hexapole (or octapole) ion guide then pulsed into the TOFanalyzer. Thus, the necessary electrostatic pulsing creates a time zero from w hich the TOF measurements canbegin.

Figure 2.10. A MALDI time-of-f light reflectron mass analyzer and its ability to generate fragmentation information.Fragmentation analysis from a MALDI TOF ref lectron is know n as post-source decay or PSD.


The MALDI w ith Time-of-Flight Analysis

In the initial stages of MALDI–TOF development, these instruments had relatively poor resolution w hichseverely limited their accuracy. An innovation that has had a dramatic effect on increasing the resolving pow erof MALDI time-of-f light instruments has been delayed extraction (DE), as show n in Figure 2.11. In theory,delayed extraction is a relatively simple means of cooling and focusing the ions immediately after the MALDIionization event, yet in practice it w as initially a challenge to pulse 10,000 volts on and off w ithin a nanosecondtime scale. In traditional MALDI instruments, the ions w ere accelerated out of the ionization source immediately asthey w ere formed. How ever, w ith delayed extraction the ions are allow ed to “cool” for ~150 nanosecondsbefore being accelerated to the analyzer. This cooling period generates a set of ions w ith a much smaller kineticenergy distribution, ultimately reducing the temporal spread of ions once they enter the TOF analyzer. Overall,this results in increased resolution and accuracy. The benefits of delayed extraction signif icantly diminish w ithlarger macromolecules such as proteins (>30,000 Da).

Figure 2.11. Delayed extraction (DE) is a technique applied in MALDI w hich allow s ions to be extracted from theionization source after a cooling period of ~150 nanoseconds. This cooling period effectively narrow s the kineticenergy distribution of the ions, thus providing higher resolution than in continuous extraction techniques.


Quadrupole Time-of-Flight MS

Quadrupole-TOF mass analyzers are typically coupled to electrospray ionization sources and more recentlythey have been successfully coupled to MALDI. The ESI quad-TOF (Figure 2.12) combines the stability of aquadrupole analyzer w ith the high eff iciency, sensitivity, and accuracy of a time-of-f light ref lectron massanalyzer. The quadrupole can act as any simple quadrupole analyzer to scan across a specif ied m/z range.How ever, it can also be used to selectively isolate a precursor ion and direct that ion into the collision cell. Theresultant fragment ions are then analyzed by the TOF ref lectron mass analyzer. Quadrupole-TOF exploits thequadrupole’s ability to select a particular ion and the ability of TOF-MS to achieve simultaneous and accuratemeasurements of ions across the full mass range. This is in contrast to conventional analyzers, such as tandemquadrupoles, w hich must scan over one mass at a time. Quadrupole-TOF analyzers offer signif icantly highersensitivity and accuracy over tandem quadrupole instruments w hen acquiring full fragment mass spectra.

The quadrupole-TOF instrument can use either the quadrupole or TOF analyzers independently or together fortandem MS experiments. The TOF component of the instrument has an upper m/z limit in excess of 10,000. Thehigh resolving pow er (~10,000) of the TOF also enables good mass measurement accuracy on the 10 ppm level.

Due to its high accuracy and sensitivity, the ESI quad-TOF mass spectrometer is being incorporated into bothproteomics and pharmacokinetics problem solving.

Figure 2.12. An electrospray ionization quadrupole time-of-f light mass spectrometer.


Fourier Transform Mass Spectrometry (FTMS)

FTMS is based on the principle of monitoring a charged particle’s orbiting motion in a magnetic f ield (Figure2.13-14). While the ions are orbiting, a pulsed radio frequency (RF) signal is used to excite them. This RFexcitation allow s the ions to produce a detectable image current by bringing them into coherent motion andenlarging the radius of the orbit. The image current generated by all of the ions can then be Fourier-transformedto obtain the component frequencies of the different ions, w hich correspond to their m/z. Because thefrequencies can be obtained w ith high accuracy, their corresponding m/z can also be calculated w ith highaccuracy. It is important to note that a signal is generated only by the coherent motion of ions under ultra-highvacuum conditions (10-11 – 10-9 Torr). This signal has to be measured for a minimum time (typically 500 ms to 1second) to provide high resolution. As pressure increases, signal decays faster due to loss of coherent motiondue to collisions (e.g. in ~ <150 ms) and does not allow for high resolution measurements (Figure 2.14).

Figure 2.13. A side view of an FTMS instrument w ith ESI source. The ESI ions are formed and guided into theanalyzer cell using a single stage quadrupole rod assembly. The analyzer cell rests in the superconductingmagnet (diagram courtesy IonSpec Corporation).

Ions undergoing coherent cyclotron motion betw een tw o electrodes are illustrated in Figure 2.13. As thepositively charged ions move aw ay from the top electrode and closer to the bottom electrode, the electric f ield ofthe ions induces electrons in the external circuit to f low and accumulate on the bottom electrode. On the otherhalf of the cyclotron orbit, the electrons leave the bottom electrode and accumulate on the top electrode as theions approach. The oscillating f low of electrons in the external circuit is called an image current. When a mixtureof ions w ith different m/z values are all simultaneously accelerated, the image current signal at the output of theamplif ier is a composite transient signal w ith frequency components representing each m/z value. In short, all ofthe ions trapped in the analyzer cell are excited into a higher cyclotron orbit, using a radio frequency pulse. Thecomposite transient image current signal of the ions as they relax is acquired by a computer and a Fouriertransform is used to separate out the individual cyclotron frequencies. The effect of pressure on the signal andresolution is demonstrated in Figure 2.14.

Figure 2.14. ESI FTMS data generated on multiple proteins, the sinusoidal composite image current for all m/zions can be Fourier transformed to measure frequencies (and therefore m/z) accurately.

In addition to high resolution, FTMS also offers the ability to perform multiple collision experiments (MSn). FTMSis capable of ejecting all but the ion of interest. The selected ion is then subjected to a collision gas (or anotherform of excitation such as laser light or electron capture) to induce fragmentation. Mass analysis can then becarried out on the fragments to generate a fragmentation spectrum. The high resolution of FTMS/MS also yieldshigh-accuracy fragment masses.

Figure 2.15. Pressure effect on transient signal and resolution.

FTMS is a relative neophyte to biomolecular analysis, yet many of its advantages are generating more andmore interest. It is now becoming more common to couple ultrahigh resolution (>105) FTMS to a w ide variety ofionization sources, including MALDI, ESI, APCI, and EI. The result of an FTMS analyzer’s high resolving pow er ishigh accuracy (often at the part per million level) as illustrated for a protein in Figure 2.15 w here individualisotopes can be observed. The Fourier transform of the ICR signal greatly enhances the utility of ICR bysimultaneously measuring all the overlaying frequencies produced by the ions w ithin the ICR cell. The individualf ’ /

frequencies can then be easily and accurately translated into the ion’s m/z.

Figure 2.16. A demonstration of deconvolution from an FTMS mass spectrum of a 10 KDa protein at a resolutionof 30,000. The cluster of peaks represents the isotope distribution of a protein and the 0.2 m/z isotope spacingindicates a 5+ charge state.

In general, increasing magnetic f ield (B) has a favorable effect on performance. The Fourier transform of theICR signal, by measuring overlaying frequencies simultaneously, allow s for high resolution and high massaccuracy w ithout compromising sensitivity. This is in sharp contrast to double sector instruments that suffer froma loss in sensitivity at the highest resolution and accuracy. The high resolution capabilities of FTMS are directlyrelated to the magnetic f ield of the FTMS superconducting magnet, w ith the resolution increasing as a linearfunction of the f ield. The ion capacity as w ell as MS/MS kinetic energy experiments increases as a square of themagnetic therefore improving dynamic range and fragmentation data. One challenge in increasing B is themagnetic mirror effect w here ion transmission to the inside of magnetic f ield becomes more diff icult due tomagnetic f ield lines. Also, manufacturing high f ield magnets w ith larger bores and excellent f ield homogeneity (inthe ICR housing) becomes technically more diff icult.

FTMS instrumentation is affected by the magnetic f ield in the follow ing w ays:

FTMS attribute Effect of Magnetic FieldStrength B

What it means:

Resolution(m/Δm)

Directly proportional to B Improves mass accuracy and the ability to getisotopic resolution on large macromolecules.

Kinetic energy Directly proportional to B2 Increases the fragmentation and also ability tofragment larger macromolecules.

Ion capacity Directly proportional to B2 Can store more ions before space-charge adverselyaffects performance.

Figure 2.17. Fourier transform ion cyclotron frequency increases w ith magnetic f ield strength. The increasedfrequency improves accuracy as it allow s for more measurements to average.

Because ion frequency = K*B*z/m, larger magnetic f ields provide a higher frequency for the same m/z,therefore more data points are generated to def ine the frequency more precisely w hich ultimately increasesaccuracy (Figure 2.17).

Quadrupole-FTMS and quadrupole linear ion trap-FTMS mass analyzers that have recently been introducedare typically coupled to electrospray ionization sources. The quad-FTMS combines the stability of a quadrupoleanalyzer w ith the high accuracy of a FTMS. The quadrupole can act as any simple quadrupole analyzer to scanacross a specif ied m/z range How ever it can also be used to selectively isolate a precursor ion and direct that

across a specif ied m/z range. How ever, it can also be used to selectively isolate a precursor ion and direct thation into the collision cell or the FTMS. The resultant precursor and fragment ions can then be analyzed by theFTMS.

Performing MS/MS experiments outside the magnet presents some advantages since high resolution in FTMSis dependent on the presence of high vacuum. MS/MS experiments involve collisions at a transiently highpressure (10-6 – 10-7 Torr) that then has to be reduced to achieve high resolution (10-10 – 10-9 Torr). PerformingMS/MS experiments outside the cell is thus faster since the ICR cell can be maintained at ultra-high vacuum. Thismakes the new er hybrid instrument designs are optimum over coupling FTMS/MS to separation techniques suchas LC.

Table 2.2. A general comparison of mass analyzers typically used for electrospray. These valuesvary with instrument manufacturer.

Quadrupole Ion Trap Time-of-Flight

Time-of-Flight

Reflectron

MagneticSector

FTMS Quadrupole-TOF

Accuracy 0.01% (100ppm)

0.01%(100ppm)

0.02 to0.2% (200ppm)

0.001% (10ppm)

<0.0005%(<5 ppm)

<0.0005%(<5 ppm)

0.001% (10ppm)

Resolution 4,000 4,000 8,000 15,000 30,000 100,000 10,000

m/zRange

4,000 4,000 >300,000 10,000 10,000 10,000 10,000

ScanSpeed

~a second ~asecond

milliseconds milliseconds ~asecond

~a second ~a second

TandemMS

MS2 (triplequad)

MSn MS MS2 MS2 MSn MS2

TandemMSComments

GoodaccuracyGoodresolutionLow -energycollisions

GoodaccuracyGoodresolutionLow -energycollisions

Notgenerallyapplicable

Precursorionselection islimited to aw ide massrange;grow ingnumber ofapplications

LimitedresolutionHigh-energycollisions

Excellentaccuracyandresolutionof productions

ExcellentaccuracyGoodresolutionLow -energycollisionsHighsensitivity

GeneralComments

Low costEase ofsw itchingpos/neg ions

Low costEase ofsw itchingpos/negionsWell-suitedMSn

Low cost GoodaccuracyGoodresolution

InstrumentismassiveCapableof highresolution

Highresolution,MSnhighvacuum,superconductingmagnet,expense

Know n forhighsensitivityand accuracyw hen usedfor MS2


Detectors

Once the ions are separated by the mass analyzer, they reach the ion detector (Figures 2.1 and 2.18-21),w hich generates a current signal from the incident ions. The most commonly used detector is the electronmultiplier, w hich transfers the kinetic energy of incident ions to a surface that in turn generates secondaryelectrons. How ever, a variety of approaches are used to detect ions depending on the type of massspectrometer.

Electron Multiplier

Perhaps the most common means of detecting ions involves an electron multiplier (Figure 2.18), w hich ismade up of a series (12 to 24) of aluminum oxide (Al2O3) dynodes maintained at ever increasing potentials. Ionsstrike the f irst dynode surface causing an emission of electrons. These electrons are then attracted to the nextdynode held at a higher potential and therefore more secondary electrons are generated. Ultimately, as numerousdynodes are involved, a cascade of electrons is formed that results in an overall current gain on the order of onemillion or higher.

Figure 2.18. Diagrammatic representation of an electron multiplier and the cascade of electrons that results in a106 amplif ication of current in a mass spectrometer.

The high energy dynode (HED) uses an accelerating electrostatic f ield to increase the velocity of the ions.Si th i l l t lti li i hi hl d d t i l it th HED t i i l

Since the signal on an electron multiplier is highly dependent on ion velocity, the HED serves to increase signalintensity and therefore sensitivity.

Faraday Cup

A Faraday cup (Figure 2.19) involves an ion striking the dynode (BeO, GaP, or CsSb) surface w hich causessecondary electrons to be ejected. This temporary electron emission induces a positive charge on the detectorand therefore a current of electrons f low ing tow ard the detector. This detector is not particularly sensitive,offering limited amplif ication of signal, yet it is tolerant of relatively high pressure.

Figure 2.19. Faraday cup converts the striking ion into a current by temporarily emitting electrons creating apositive charge and the adsorption of the charge from the ion striking the detector.

Photomultiplier Conversion Dynode

The photomultiplier conversion dynode detector (Figure 2.20) is not as commonly used at the electronmultiplier yet it is similar in design w here the secondary electrons strike a phosphorus screen instead of adynode. The phosphorus screen releases photons w hich are detected by the photomultiplier. Photomultipliersalso operate like the electron multiplier w here the striking of the photon on a scintillating surface results in therelease of electrons that are then amplif ied using the cascading principle. One advantage of the conversiondynode is that the photomultiplier tube is sealed in a vacuum, unexposed to the environment of the massspectrometer and thus the possibility of contamination is removed. This improves the lifetimes of these detectorsover electron multipliers. A f ive-year or greater lifetime is typical, and they have a similar sensitivity to theelectron multiplier.

Figure 2.20. Scintillation counting w ith a conversion dynode and a photomultiplier relies on the conversion of theion (or electron) signal into light. Once the photon(s) are formed, detection is performed w ith a photomultiplier.

Array Detector

An array detector is a group of individual detectors aligned in an array format. The array detector, w hichspatially detects ions according to their dif ferent m/z, has been typically used on magnetic sector massanalyzers. Spatially dif ferentiated ions can be detected simultaneously by an array detector. The primaryadvantage of this approach is that, over a small mass range, scanning is not necessary and therefore sensitivityis improved.

Charge(or Inductive) Detector

Charge detectors simply recognize a moving charged particle (an ion) through the induction of a current onthe plate as the ion moves past. A typical signal is show n in Figure 2.21. This type of detection is w idely used inFTMS to generate an image current of an ion. Detection is independent of ion size and therefore has been usedon particles such as w hole viruses.

Figure 2.21. Illustration of the operation of a mass spectrometer w ith a charge detector; as a charged speciespasses through a plate it induces a current on the plate.

Table 2.3. General comparison of detectors.

Detector Advantages Disadvantages

g g

Faraday CupGood for checking iiontransmission and low sensitivitymeasurements

Low amplif ication (≈10)

PhotomultiplierConversion Dynode(ScintillationCounting)

RobustLong lifetime (>5 years)Sensitive (≈gains of 106)

Cannot be exposed to light w hilein operation

Electron MultiplierRobustFast responseSensitive (≈gains of 106)

Shorter lifetime than scintillationcounting (~3 years)

High EnergyDynodes w ithelectron multiplier

Increases high mass sensitivity May shorten lifetime of electronmultiplier

ArrayFast and sensitive Reduces resolution

Expensive

Charge DetectionDetects ions independent of massand velocity

Limited compatibility w ith mostexisting instruments


Vacuum in the Mass Spectrometer

All mass spectrometers need a vacuum to allow ions to reach the detector w ithout colliding w ith othergaseous molecules or atoms. If such collisions did occur, the instrument w ould suffer from reduced resolutionand sensitivity. Higher pressures may also cause high voltages to discharge to ground w hich can damage theinstrument, its electronics, and/or the computer system running the mass spectrometer. An extreme leak,basically an implosion, can seriously damage a mass spectrometer by destroying electrostatic lenses, coating theoptics w ith pump oil, and damaging the detector. In general, maintaining a good vacuum is crucial to obtaining highquality spectra.

One of the f irst obstacles faced by the originators of mass spectrometry w as coupling the sample source to amass spectrometer. The sample is initially at atmospheric pressure (760 torr) before being transferred into themass spectrometer’s vacuum (~10-6 torr), w hich represents approximately a billion-fold difference in pressure.One approach is to introduce the sample through a capillary column (GC) or through a small orif ice directly intothe instrument. Another approach is to evacuate the sample chamber through a vacuum lock (MALDI) and once areasonable vacuum is achieved (< 10-2 torr) the sample can be presented to the primary vacuum chamber (< 10-

5 torr).

A mass spectrometer is show n in Figure 2.22 w ith three alternative pumping systems. All three systems arecapable of producing a very high vacuum, and are all backed by a mechanical pump. The mechanical pumpserves as a general w orkhorse for most mass spectrometers and allow s for an initial vacuum of about 10-3 torrto be obtained. Once a 10-3 torr vacuum is achieved, the other pumping systems, such as diffusion, cryogenicand turbomolecular can be activated to obtain pressures as low as 10-11 torr.

Figure 2.22. A w ell-maintained vacuum is essential to the function of a mass spectrometer. A couple of thedifferent types of vacuum systems are illustrated.


Summary

The mass analyzer is a critical component to the performance of any mass spectrometer. Among the mostcommonly used are the quadrupole, quadrupole ion trap, time-of-f light, time-of-f light ref lectron, and FTMS.How ever, the list is grow ing as more specialized analyzers allow for more diff icult questions to be addressed.For example, the development of the quad-TOF has demonstrated its superior capabilities in high accuracytandem mass spectrometry experiments. Once the ions are separated by the mass analyzer they reach the iondetector, w hich is ultimately responsible for the signal w e observe in the mass spectrum.

References

Busch K.L., Glish G.L., McLuckey S.A. Mass Spectrometry/Mass Spectrometry: Techniques and Applications ofTandem. John Wiley & Sons, 1989.

Cotter R. Time-Of-Flight Mass Spectrometry: Instrumentation and Applications in Biological Research.Washington, D.C.: ACS, 1997.

McCloskey J.A. & Simon M.I. Methods in Enzymology: Mass Spectrometry. Academic Press, 1997.

Kinter M. & Sherman NE. Protein Sequencing and Identification Using Tandem Mass Spectrometry. Wiley-Interscience, 2000.


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