Matrix-assisted laser desorption/ionisation–mass spectrometry applied to biological macromolecules

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Matrix-assisted laser desorption /ionisation^mass spectrometry applied tobiological macromoleculesJulia Gross, Kerstin Strupat*Institute for Medical Physics and Biophysics, University of Muë nster, Robert-Koch Strasse 31,D-48149 Muë nster, Germany

Since its invention in the late 1980s, matrix-assisted laser desorption / ionisation^massspectrometry (MALDI-MS) has been appliedto a large number of analytical problemssuch as the analysis of proteins, glycoconju-gates, polynucleotides and synthetic poly-mers. This article explains brie£y the principleof MALDI and the instrumentation required.The preparation and puri¢cation of sampleson a microlitre scale prior to mass analysisare described. Examples are given of MALDImass analysis using infrared and ultravioletlasers ( IR^ and UV^MALDI-MS). Analyticalapplications of MALDI-MS to mass analysisof electroblotted proteins, to unambiguousprotein identi¢cation, the de novo sequencingof polynucleotides, DNA mutant analysis fordiagnostic purposes, and the analysis of non-covalent complexes, are addressed. z1998Elsevier Science B.V. All rights reserved.

Keywords: Matrix-assisted laser desorption / ionisation^massspectrometry; Lasers; Sample puri¢cation; Proteins,oligosaccharides, oligonucleotides; Electroblotting;Non-covalent complexes: Gel electrophoresis

1. Introduction

The invention and development of the so-called softionisation techniques, secondary ion mass spectrom-

etry (SIMS), fast atom bombardment^mass spectrom-etry (FAB-MS), plasma desorption mass spectrome-try (PDMS), and the need for mass analysis of bio-logically relevant compounds, resulted in thedevelopment of electrospray-ionisation^mass spec-trometry (ESI-MS) and matrix-assisted laser desorp-tion / ionisation^mass spectrometry (MALDI-MS)[ 1^5 ]. Both ESI- and MALDI-MS, established inthe late 1980s, show extremely high sensitivity andan excellent accuracy of mass determination for themass analysis of very high mass compounds such aspeptides, proteins, glycoconjugates, oligonucleotides,and synthetic polymers. The following discussionrefers only to MALDI-MS results.

The sensitivity of the MALDI techniques ( i.e.amount of sample required per preparation) is in thesub-picomole ^ and quite often even in the low fem-tomole ^ range. Under UV^MALDI conditions, onlyattomoles or sub-attomoles are consumed per lasershot [ 6,7 ]. The accessible mass range of ( the classi-cally used) time-of-£ight mass analysers allows one todetect analyte molecules having m / z values of 300kDa and higher. Using delayed ion extraction the pre-cision of mass determination amounts to approxi-mately 5^10 ppm, and its accuracy is 2^5 ppm formasses below 5 kDa; accuracies in the 50^200 ppmrange can be achieved for masses above 25 kDa [ 8,9 ].Typically, in the mass spectrum the signal of an intactmolecular ion is predominantly detected as the singlycharged species. Singly ^ and more highly ^ chargedoligomers and monomers are detected at the corre-sponding m /z ratios. The desorption / ionisation proc-ess which is induced, as well as the matrix-inducedfragmentation of analytes ( smaller than ca 3^4 kDa),can be used for sequencing. In-source decay^MALDI-MS (ISD-MALDI-MS) [ 10 ] is based on speci¢c ana-lyte fragmentation in the ion source. In contrast, post-source decay^MALDI-MS (PSD-MALDI-MS) [ 11]uses speci¢c fragmentations which occur in the ¢eld-free drift region of the time-of-£ight mass analyser.

0165-9936/98/$ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 5 - 9 9 3 6 ( 9 8 ) 0 0 0 6 0 - 0

*Corresponding author.

Abbreviations: UV- and IR-MALDI^MS, ultraviolet- andinfrared-matrix-assisted laser desorption/ionisation^massspectrometry; TOF, time-of-flight; PSD, post-source decay;ISD, in-source decay; DE, delayed extraction; nt, nucleo-tides; SDS, sodium dodecyl sulfate; 2D-PAGE, two-dimen-sional polyacrylamide gel electrophoresis ; PVDF,polyvinylidene fluoride

470 trends in analytical chemistry, vol. 17, nos. 8+9, 1998

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The number of papers published in the last threeyears dealing with MALDI-MS amounts to approxi-mately 2500, and it is obvious that not all of the manydifferent aspects of, or compounds analysed by,MALDI can be discussed. This article does not dealwith carbohydrates, glycoconjugates, lipids (and theirconjugates ) or synthetic polymers: for more informa-tion on these, see [ 12,13 ]. Our purpose is to offer asummary of the analytical `state of the art' of MALDI-MS. Details of the instrumentation, and the basics ofmass spectrometry, are not covered: they may befound elsewhere [ 14,15 ].

The principal idea in MALDI preparations is toobtain a mixture of low concentration analyte mole-cules (1034^1037 M ) and high concentration matrixmolecules (1031 M ) on the sample support, resultingin a large molar excess of the matrix. The matrix con-sists of small organic compounds which show a strongresonance absorption at the applied laser wavelength,and is therefore responsible for the energy depositioninto the matrixânalyte mixture. Under UV^MALDIconditions the matrix molecules are excited electroni-cally, whereas vibrational excitation takes place in IR^MALDI. Pulsed laser systems (UV lasers, with a laser-pulse duration d = 0.5^20 ns, or IR lasers, d = 5^200ns) are used to enable an explosive disintegration of alaser-light-excited matrixânalyte volume, and thus tothe desorption with ionisation. Various desorption /ionisation models have been discussed for UV-MALDI-MS [ 16^19 ], but it should be noted that theprocess itself is far from well understood. This holdstrue even more for the IR^MALDI process. Besidesthe role of the matrix being to absorb the laser light andsupporting or inducing the ionisation of analyte mol-ecules, its high molar excess provides separation ofanalyte molecules from each other and, therefore,reduces the intermolecular interactions between indi-vidual analyte molecules.

2. Instrumentation

2.1. Mass spectrometers

MALDI-MS has been performed using varioustypes of mass analysers [ 2^4,20^23 ]. Themost straightforward approach uses the couplingwith time-of-£ight (TOF) mass analysers. In thelast 10 years, MALDI has been coupled to both linearand re£ectron TOF systems, and the latest resultsreport the successful coupling of MALDI with FTÎCR mass analysers and with quadrupole-TOF sys-

tems (Q-TOF), using orthogonal extraction condi-tions.

One of the most important improvements inMALDI-TOF was the application of delayed ionextraction (as delayed-ion extraction, DE-MALDI),shown by a number of research groups [ 24^27 ]. Theprincipal idea of delayed ion extraction had been pub-lished by Wiley and McLaren in the 1950s [ 28,29 ].DE-MALDI results show highly enhanced mass reso-lution, in linear as well as in re£ectron OF-systems.

2.2. Lasers

Since MALDI was invented after studies of theinteraction of UV-laser light with small organic com-pounds, which provide differing extinction coef¢-cients [ 30 ], the ¢rst lasers had a wavelength rangebelow 300 nm, where small aromatic molecules,such as aromatic amino acids, have high resonantabsorption. Therefore, a laser of wavelength 266 nm(frequency-quadrupled Nd-YAG laser ) was appliedto MALDI-MS by Karas and Hillenkamp, using nic-otinic acid as a matrix to detect proteins with molec-ular masses exceeding 10 000 Da [ 31]. Later, matriceswere found for 337 nm (N2 laser ) and 355 nm (fre-quency-tripled Nd-YAG laser ) [ 32^34 ]. All the dif-ferent UV-laser wavelengths have been applied suc-cessfully to MALDI-MS. However, N2 laser systemsare used routinely in most laboratories, since they arereliable and inexpensive. In a parallel development,lasers in the infrared wavelength range have beenapplied. Systems emitting close to 3 Wm (2.79 Wm,Er-YSSG laser; 2.94 Wm, Er-YAG laser ) [ 35,36 ],and 10 Wm (CO2 laser ) [ 37 ] were tested, and provedthe principle feasibility of this wavelength range withMALDI. Recently, the ¢rst investigations of thedesorption / ionisation process using a tuneable freeelectron laser system [ 38 ] and OPO systems[ 39,40 ] have been published.

3. Sample preparation for MALDI

As described above, MALDI-MS is an effectivetechnique for mass analysis of various classes of com-pounds. For a successful MALDI mass analysis it isimportant to choose a proper matrix for the individualclass of analyte combination with a suitable method ofpreparation. The key to a fruitful MALDI analysis liesin the preparation technique applied to the analyticalproblem as well as the choice of appropriate matrix,analyte, and wavelength combinations.

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A routinely used preparation procedure uses thedried-droplet method in combination with the matri-ces 2,5-dihydroxybenzoic acid (DHB) [ 6 ], alone ortogether with additives ( superDHB which is 2-hydroxy-5-methoxy-benzoic acid ) [ 41], succinicacid (only for an IR matrix ) for peptides, proteinsand carbohydrates, or 3-hydroxypicolinic acid (3-HPA) [ 42 ] for oligonucleotide analysis. About 2 Wlof aqueous solutions of DHB, superDHB, or succinicacid (all 15^25 g / l ) or 3-HPA (50 g / l ), are mixed on-target with 0.5 Wl of an aqueous solution of the analyte,and are allowed to dry in a gentle stream of air. Thesematrices show a somewhat more heterogeneous sam-ple morphology, and therefore induce a lower accu-racy of mass determination ^ especially pronounced inlinear TOF mass analysers ^ than do the homogeneoussample preparations described next.

The thin-layer preparation, or so-called fast evap-oration technique, is performed with matrices havinglow solubility in water, such as K-cyano-4-hydroxy-cinnamic acid (ACCA) [ 43,44 ] or sinapinic acid(SA). The matrix solution, dissolved in high concen-tration (up to saturation) in a volatile solvent such asacetone, is applied on the sample support. The aqueousanalyte solution is strati¢ed onto this layer and, owingto the low water solubility of the matrix, the matrixlayer is not redissolved. The addition of nitrocellulose(NC) to an ACCA matrix has proved to be very effec-tive. Nitrocellulose, used as a matrix additive, isbelieved to be responsible for a higher tolerancetowards salts and buffers [ 45,46 ]. Increasing thenitrocellulose concentration results in thick layers(`thick-layer preparations') and higher impurity con-centrations can be tolerated (see Section 4). Unfortu-nately, the ACCA matrix mostly used for thin-layerpreparations induces many metastable fragmentationsof the analyte molecules [ 47 ], and for this reason it isnot the matrix of choice for high mass compoundsabove ca 15 kDa measured in a re£ectron TOF system[ 47 ].

4. Sample puri¢cation

In addition to the choice of matrix and sample prep-aration, the chemical environment of the analyte is ofvital importance. Although MALDI has a relativelyhigh tolerance towards impurities such as detergents,buffers, or other sample contamination, the mass spec-trometric analysis may be impaired by a disturbedcrystallisation of the matrix, high salt concentrationsin the preparation, and peak broadening by fragmen-

tation and /or adduct formation. All of these can resultin a strong reduction of signal intensity ( i.e. low sig-nal-to-noise ratios ). The upper limit for the concen-tration of different buffers varies within the 10^100millimolar range, so sample dilution may suf¢ce forachieving good spectra of analyte molecules. Phos-phate buffers complicate the analysis and Tris^HClbuffers should be used instead. In general, zwitterionicdetergents such as CHAPS or SDS cannot be accepted,and have to be exchanged for non-ionic detergentssuch as octylglycoside or Triton X-100 [ 48 ]. The tol-erance of MALDI towards contaminants relies on thecrystallisation process of the matrix and analyte, andcan be described as on-target chromatography (puri¢-cation by crystallisation). Impurities are excluded in atime-dependent manner, from the forming of earlycrystals on the rim, towards the amorphous area inthe middle of the sample spot. This argument holdstrue for succinic acid, and some benzoic acid and pico-linic acid derivatives. For UV^MALDI and mostmatrices it has become clear that the use of glycerolas solvent should be avoided since it prevents com-plete matrix crystallisation. On the other hand, glyc-erol is one of the most favoured IR matrices ( see later ).At least under UV^MALDI conditions, the solventDMSO has to be avoided, even in traces, since it dis-turbs crystallisation.

Many different puri¢cation approaches have beendeveloped which allow one to choose the optimal pro-tocol, depending on the nature of the sample. The aimis to develop simple methods that allow rapid puri¢-cations, do not waste material, and can be applied formost classes of analytes.

Organic ammonium salts ( citrate, tartrate, or ace-tate ) can be added to the matrix prior to or duringsample preparation, and are found to enhance signalintensities. Whether this effect is based on suppressionof alkali-adduct formation [ 49 ], improved incorpora-tion of the analyte in the matrix crystals [ 50 ], or / andon a more effective ionisation /desorption process[ 51], is still being discussed. By using the thin- orthick-layer preparation mentioned above, cationscan be removed from the ¢nally prepared sample[ 43,44 ]. This approach relies on the low solubilityof ACCA or SA in water ^ which is in contrast toinorganic salts, which appear on top of the preparedsamples. The impurities are redissolved in a fewmicrolitres of ice-cold water, pipetted onto the samplepreparation, and then the water is removed after a fewminutes [ 43,44 ]. Another simple approach for remov-ing cations uses cation exchange polymer beads (Bio-Rad), in their protonated or ammonium form. These


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beads (50W-X8, mesh size 100^200 Wm) can beapplied for cation exchange in the analyte and /ormatrix solution [ 52 ], as well as in the aqueous prep-aration on the target ( to cope with cations of the metaltarget ) [ 53 ]. Exchange of buffers, or removal of deter-gents and other low weight impurities, can be per-formed with dialysis on membrane ¢lters (Millipore ).Dialysis with sample volumes of a few microlitres iscarried out for 15^20 min on a membrane £oating onthe exchange buffer (which might also be neat water ).A clean-up procedure based on micro-reversed-phasechromatography, which enables the separation ofmolecules belonging to one analyte class, was intro-duced by Kussmann et al. [ 54 ]. The separation is per-formed in a GelLoader Tip (Eppendorf ) with Porosmaterial (PerSeptive Biosystems) as the stationaryphase (1^5 Wl, applied in methanol ). The separationof the analyte molecules from contaminants can beachieved rapidly in one chromatographic step. Theanalyte is eluted directly onto the MALDI target.

5. Applications of UV^ andIR^MALDI-MS

5.1. Protein identi¢cation by tryptic digestionand tag sequencing

The identi¢cation of a protein relies on an unambig-uous response from searches of protein database( s )which provide protein-speci¢c information about theindividual protein under investigation. This informa-tion includes the mass of the intact protein, its confor-mation, speci¢c chemical or enzymatic fragments, orpartial sequence information on the protein. The num-ber of proteins accessible using protein databases [ 55 ]is approximately 220 000, taking into account differ-ent organisms. Since MALDI provides mass analysesof peptides, relatively independently of their individ-ual features, and shows a high tolerance towards com-monly used buffers, the enzymatic treatment of pro-teins followed by mass analysis of the speci¢cproteolytic fragments provides a very straightforwardapproach. The principal strategy for identifying pro-teins with the aid of protein databases is, ¢rst, to digestthe protein using speci¢c proteases, either in solution,in gel or on membrane [ 56^60 ], and then to mass-analyse the proteolytic fragments, after suitable prep-aration techniques. The mass analysis of these protein-speci¢c fragments (`¢ngerprint') provides valuableinformation on the individual protein, and databasescan be searched using a number of search algorithms

which are available on the Internet. These includehttp: / /prospector.ucsf.edu /htmlucsf / ms¢t.htm,http: / /prowl.rockefeller.edu /cgi-bin /ProFound, andhttp: / /www.mann.embl-heidelberg.de /Services /Pe-ptideSearch /FR^PeptideSearchForm.html.

The reliability of protein identi¢cation by MALDImass analysis has improved dramatically since theintroduction of DE-MALDI-MS [ 61]. An exampleof this approach using the high accuracy of DE^MALDI is shown in Fig. 1, for the protein phospho-fructokinase. However, it must be noted that manyreal-life samples cannot be identi¢ed unambiguouslyfrom the proteolytic fragments alone. Therefore, a sec-ond analytical step is required to provide informationon part of the amino acid sequence of one or more ofthe proteolytic fragments. The idea of `tag sequenc-ing' was ¢rst published by Wilm and Mann for colli-sion-induced fragmentation using ESI-MS [ 62 ]. Apartial amino acid sequence of an individual proteo-lytic fragment can be derived from speci¢c fragmen-tations taking place either in the ion-source ( ISD) or inthe ¢eld-free drift tube (PSD) of the mass spectrom-eter. Again, several search algorithms are available,with links to protein databases (http: / /www.mann.embl-heidelberg.de /Services /PeptideSearch /FR^PeptidePatternForm.html ).

It should be noted, however, that de novo sequenc-ing of peptides or proteins is still far from being routinewith MALDI-MS. Since the fragmentation rules andspeci¢city are better understood and /or more reliableunder ESI-CID conditions, it is suggested that de novosequencing be performed by ESI-MS [ 63 ].

6. MALDI-MS of polynucleotides

The quality of performance of mass spectrometricanalysis of oligo- and polynucleotides has increasedrapidly as a result of optimised preparation and puri¢-cation methods. In general, the upper mass limit forDNA is about 90 kDa (ca 300-mer ) [ 64 ], and forRNA, because of its higher stability in the MALDIprocess, about 150 kDa (ca 500-mer ) [ 65 ] with asensitivity that can be increased to the femtomolerange for both subclasses. The accessible mass rangeis determined by the susceptibility of oligonucleotidesto fragmentation and the high af¢nity to metal-ionadduct formation. Peak broadening caused by meta-stable decay of the analyte, and heterogeneous adductformation, reduces the signal intensities and resolu-tion. The fragmentation depends on the analyte andon a combination of the applied matrix and laser wave-


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length, and increases with the mass of the analyte ion[ 66^68 ]. The introduction of new matrices and addi-tives, as well as the employment of modi¢ed bases orsugars, has led to a reduced fragmentation yield. Theuse of cation exchange polymer beads and the additionof ammonium salts as matrix additives provide effec-tive methods for enhancing the detection of non-cat-ionised ion species. The use of 3-HPA in combinationwith a laser wavelength of 337 nm provides a signi¢-cantly reduced fragmentation yield, a better signal-to-noise ratio, and comparable signal intensities for boththe negative and positive ion mode [ 69 ]. Combina-tions of 3-HPA with different additives such as pico-linic acid (PA) (at 266 nm [ 70 ] or at 337 or 355 nm[ 71]), or ammonium citrate, are used for the detectionof larger DNAs. Zhu et al. demonstrated the feasibili-ty of detecting DNA up to a 150-mer with little as1 pmole of material prepared with a matrix combina-tion of 2,3,4- and 2,4,6-trihydroxyacetophenone andammonium citrate [ 72 ]. However, the mass resolutionand signal-to-noise ratio are very low in this massrange and for this kind of preparation. In general,DNA shows a mass resolution of only 20^60FWHM (full width at half maximum) for UV^MALDI-MS with more than 80 nt [ 53,69,70 ]. The

introduction of delayed ion extraction led to a dramat-ically increased mass resolution, higher signal-to-noise ratio, and increased detection sensitivity up toDNA 50-mers. The result was achieved as a result ofhigher instrumental resolving power and reduced frag-mentation in the initial ¢eld-free expansion of the gen-erated plume using DE conditions [ 73^75 ]. Moredetails are given in the review by Nordhoff et al.,which describes the analysis of DNA /RNA withMALDI-MS and ESI-MS [ 76 ].

Sequence information is important for the charac-terisation of analytes and can be deduced fromMALDI-induced fragments and enzymatic or chemi-cal reactions. Sequencing via prompt fragmentsinduced by IR^MALDI was demonstrated by Nord-hoff et al. for a DNA 21-mer in the negative ion mode[ 77 ]. Dif¢culties arose, however, with low shot-to-shot reproducibility of the laser, high metastabledecay of the analyte, and the reduced fragmentationyield at thymine sites. Structural information can alsobe gained by using PSD-UV^MALDI-MS. Althougheven thymine bases can be protonated and cleaved, themuch lower fragmentation of thymidines, multiplebase losses, and multiple pathways hamper the inter-pretation of the PSD spectrum [ 78,79 ].

Fig. 1. Certain identi¢cation of a 108-kDa protein (yeast phosphofructokinase, SwissProt P16861) by database search usingonly eight peptide masses. The mass accuracies are indicated above the matching peaks. Signals at the m /z = 1060.10( matrix-related ion signal ) and 2163.057 ( trypsin autodigestion peptide) were used for internal calibration. From [ 61], withpermission.


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Exonucleases which cleave speci¢cally fromone terminus and release successive mononucleotidescan be used very ef¢ciently for unambiguous sequenc-ing. The time-dependent digestion by the exonu-cleases allows one to obtain sequence ladders, owingto the individual enzyme kinetics. One digest reactionis therefore suf¢cient to measure small aliquots at

different times. Smirnov et al. demonstrated theapplicability of this approach and its extension up to50 nt by using delayed ion extraction and optimisingthe enzymatic digestion conditions [ 80 ]. Fig. 2shows the spectrum of the digest of a DNA 33-merwith snake venom and calf spleen phosphodiester-ase.

Fig. 2. Comparison of partial 3PC5P and 5PC3P exonuclease digestion analysed by delayed ion extraction UV^MALDI-MS.The time of each digestion is indicated. Both peak 33A and peak 1G correspond to the undigested 33-mer of sequence 5P-GCCAGG GTT TTC CCA GTC ACG ATG CAG AAT TCA-3P. (a ) 3PC5P digestion with snake venom phosphodiesterase;( b) 5PC3P digestion with calf spleen phophodiesterase. From [ 80 ], with permission.


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Sequencing RNA by using the above describedapproach is dif¢cult since the mass difference betweenC and U is only 1 Da. One research group is reporting alower cleavage rate of cytidine sites and, therefore,enhanced signal intensities for these fragments, gen-erated with bovine spleen phosphodiesterase [ 81], butit still has to be proved that this observation is repro-ducible for a variety of base compositions and analyteswith varying length. The problem of the small massdifference between U and C can be overcome by usingbase-speci¢c endonucleases. Hahner et al. demon-strated this solution by parallel digestion of a syntheticRNA 25-mer with different RNases and limited alka-line hydrolysis [ 82 ]. The current mass range of thisapproach is estimated to be above 100 nt.

Because of the possibility of non-speci¢c reactionsof endonucleases under conditions compatible withMALDI-MS, Tolson and Nicholson have combinedenzymatic with chemical cleavages to gain sequenceinformation by applying a C-speci¢c cleavage of syn-thetic RNA with hydrazine /aniline [ 83 ].

The detection of Sanger sequencing ladders can beachieved with MALDI-MS instead of gel electropho-resis. It is also possible to use this combination,together with the polymerase chain reaction, toamplify the DNA of interest. The coupling of DNAto solid phases allows extensive washing, with theresult that all distracting compounds such as mononu-cleotides, buffers, salts and enzymes, can be removed.Streptavidin-coated magnetic beads which interactwith biotinylated DNA can function as a solid support.Only the non-immobilised strand is detected by usingMALDI-MS [ 84 ]. Many different approaches for theidenti¢cation of mutation sites or polymorphicsequences of known DNA loci are discussed in theliterature [ 64,85 ]. Diagnostic sequencing of DNAcan be performed by immobilisation of templateDNA on streptavidin-coated magnetic beads andhybridisation of the primer. Primer extension in thepresence of dideoxynucleotides results in sequenceladders. The feasibility of this method was shown byKoëster et al. for a synthetic 78-mer [ 86 ]. A similarapproach enables rapid de novo sequencing of DNA.A duplex probe, meaning double-stranded DNA withone strand immobilised to a solid phase and the com-plementary strand possessing a 3P ¢ve-base single-stranded overhang, is used to capture unknown DNAand the sequencing is performed with a primer exten-sion reaction. This sequencing procedure was pre-sented for a DNA 31-mer [ 86 ]. The employment of7-deazapurines for the extension reaction provided anincreased stability of the generated fragments.

7. MALDI-MS of non-covalentcomplexes

Following the invention of MALDI-MS attemptshave been made to study speci¢c non-covalent pro-tein^protein or protein^ligand interactions. In com-parison to the large number of ESI-MS results reported(see the review by Loo [ 87 ]), there are only a fewreports on this MALDI topic. This is certainly a resultof the facts that it is essential to prepare the samplesproperly and to choose appropriate matrices forMALDI mass analysis. A review of MALDI resultson non-speci¢c complexes, and a discussion of theprerequisites, has been given recently by Hillenkamp[ 88 ].

In order to evaluate properly MALDI results onnon-covalent complexes, an important aspect of typi-cal MALDI mass spectra needs to be kept in mind.This is that there can be non-speci¢c oligomerisationof most of the likely gas-phase reactions induced bythe MALDI process. More speci¢cally, non-speci¢ccomplex formation between two proteins, for exam-ple, cytochrome c and myoglobin, is very easilyobtained under typical MALDI conditions but doesnot represent a speci¢c non-covalent complex. Whentypical molar matrix-to-analyte ratios are used theabundances of these oligomer signals are much lessintense than the singly charged ( monomeric ) proteins.Therefore, it should be required that the signal inten-sities at m /z ratios corresponding to the quaternarystructure (e.g. a tetramer signal ) are comparable tothe signal intensity at m /z ratios corresponding tothe monomer, and are much more intense than signalscorresponding to unspeci¢c oligomers (e.g. trimer orpentamer signals ).

The feasibility of detecting intact non-covalentlybound protein subunits was ¢rst described in 1989and 1990. Using nicotinic acid and the laser wave-length 266 nm, Karas et al. [ 89,90 ] showed the detec-tion of the trimer of the membrane protein, porin (112kDa), and of the tetramer of glucose isomerase (172kDa). Later, Rosinke et al. reported that the quaternarystructures of streptavidin (52 kDa) and porin could bedetected using a wavelength of 355 nm [ 91]. Theyreported that the intact non-covalent complex in qua-ternary protein structures was observed only for the¢rst shot onto a given spot: subsequent shots onto thisspot resulted in the detection of the monomers as thebase peak in the spectrum. Cohen et al. investigatedthis behaviour more systematically and observed it forother proteins consisting of subunits [ 92 ]. Fig. 3shows the mass spectra obtained for streptavidin


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using 2,5-dihydroxybenzoic acid (Fig. 3a ) ^ a matrixnot suitable for the detection of ( intact ) quaternarystructures ^ and 2,6-dihydroxyacetophenone, one ofthe suitable matrices. In the latter case, the ¢rst shotsonto a given spot (Fig. 3b) as well as the followingshots onto the same spot (Fig. 3c ) are shown. Possible

explanations for the observation of the `¢rst shot phe-nomenon' are discussed by Cohen et al. [ 92 ]. Thisbehaviour was also observed later by van Dorsselaer'sgroup [ 93 ].

Reichenbecher and his co-workers reported thedetection of a heterodimer of the enzyme transhydrox-

Fig. 3. UV^MALDI mass spectra of streptavidin monomer and tetramer using different matrices. Sum of 10 single-shot spectra.Laser wavelength: 355 nm. (a) 2,5-Dihydroxybenzoic acid matrix. Gas-phase reactions between monomers lead to unspeci¢coligomerization. The ¢rst and following shots of given spots show the same principal mass spectra. ( b) 2,6-Dihydroxyace-tophenone matrix. Summation spectrum of ¢rst shots onto given spots only. The signal for the speci¢c tetramer of streptavidinis the base peak in the spectrum. (c ) 2,6-Dihydroxyacetophenone matrix. Summation spectrum of second and following shotsonto given spots only. Gas-phase reactions between monomers lead to unspeci¢c oligomerization. From [ 92 ], with permis-sion.


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ylase using SA as matrix [ 94 ]. Varying the pH of thematrixânalyte solution resulted in the observation ofthe heterodimer or heterotetramer, respectively.

To date, there are only few examples of the detec-tion of intact protein^ligand complexes or protein^metal-ion complexes: Woods et al. have describedthe observation of metal-binding complexes andnon-covalent proteinênzyme complexes up to 30kDa. In these investigations ACCA and SA, respec-tively, were used as matrices, together with ammo-nium salts, in order to reduce the matrix acidity insolution [ 95 ]. The same approach was followed byGlocker and his co-workers, who reported on theRNase S^S peptide complexation. The non-covalentstructure was observed using 6-azathiothymine (6-ATT) as matrix, plus ammonium salts [ 96 ]. Recently,Gruic-Sovulj et al. reported the observation of twodifferent aminoacyl-tRNA synthetase / tRNA com-plexes using 6-ATT as matrix [ 97 ]. The speci¢cityof complex formation was proved by competitionexperiments.

Although the `¢rst shot phenomenon' has not beenreported for all non-covalent complexes, it is believedthat the key to a successful MALDI mass analysis ofnon-covalently bound compounds is the understand-ing of the sample preparation. Future research, there-fore, needs to apply appropriate analysis techniques inorder to gain a better understanding of the matrixânalyte interaction on a nanometre scale, and todescribe the matrixânalyte interaction on top of,and /or inside the matrix crystals. The detection ofspeci¢c non-covalent complexes and the related deter-mination of dissociation constants ^ by mass spec-trometry, in general, and by MALDI-MS in particular^ is one of the most important goals for the future,since life strictly depends on the binding of, andrelease from, partners in order to start or stop processes( signalling) in biological systems.

8. MALDI^MS with lasers in the infraredwavelength range ( IR-MALDI^MS)

Only some years after the invention of UV^MALDI-MS were infrared lasers applied successfullyto MALDI [ 35^37 ]. Overberg and his co-workersapplied Er-YSGG, Er-YAG and CO2 laser radiationusing aliphatic and aromatic mono- and dicarboxylicacids such as the common UV matrices, or succinicacid (SucAc), glycerol, HCl and urea to MALDI. Thespectral quality of the results obtained was the same as,or very similar to, UV^MALDI. Typically, singly

charged ion species are the base peaks in the IR^MALDI mass spectra, as in UV. For both laser wave-length ranges a more or less pronounced distribution tosingly charged oligomers and more highly chargedmonomers can be observed, depending on thematrix-to-analyte molar ratio in the sample. The inten-sities of the analyte signals obtained by IR-MALDI aretypically a factor of 1^5 higher than by UV, and it is afeature of IR^MALDI-MS that it produces less frag-mentation of analyte molecules [ 98 ]. The latter fact isobvious from the much steeper shoulders to the lowermass side of analyte signals under IR^MALDI condi-tions [ 98 ]. The higher stability of analyte moleculesunder IR^MALDI conditions makes it easier to obtainvery high mass molecules and to pro¢t from re£ectronTOF systems and the much higher achievable massresolution [ 98 ]. Berkenkamp et al. reported on thedetection of gramicidin s synthetase (510 kDa) byIR^MALDI and, principally, the much larger accessi-ble mass range available by IR^MALDI could beshown by the detection of large oligomers of a mono-clonal antibody. Clearly, a signal of the triply charged13-mer of the antibody at m /z = 650 kDa, correspond-ing to a molecular mass of the desorbed or ( morelikely ) gas-phase-induced oligomer of 1.95 MDa,could be detected using a conventional post-acceler-ation detector.

It should be noted that the desorption / ionisationprocess in IR^MALDI is far from being understood.Since the photon energies provided by the IR lasers aremuch too low (hX = 0.4 eV at 3 Wm; hX = 0.1 eV at 10.6Wm), an ionisation process via photoionisation, as pro-posed by Ehring et al. [ 99 ] for UV^MALDI, is notpossible. Recently, Cramer and co-workers discussedthe use of the `spallation model' to explain the desorp-tion / ionisation process in IR^MALDI-MS [ 38 ]. Niuet al. [ 40 ] proposed a phase transition model toexplain the desorption / ionisation process for UVând IR^MALDI, generally.

It is obvious from microscopic inspection that thesample desorption per laser shot is much larger ( by afactor of 100) in IR^MALDI than in UV^MALDI, butmore systematic studies, using large grown singlecrystals of succinic acid, were necessary to determineabsolute values. These studies resulted in the determi-nation of the absolute amount of desorbed analytemolecules per laser shot of 1^5 fmole (compared toatto- or sub-attomole amounts in UV^MALDI)[ 100 ]. According to today's understanding of theIR^MALDI process, much of the desorbed materialis not desorbed in molecular pieces, but as large unspe-ci¢c particles. It was expected from the much greater


TRAC 2481 8-9-98

desorbed sample per laser shot that the physical chem-istry in the expanding plume in IR^MALDI should bevery different from that in UV^MALDI [ 100 ]. How-ever, the ¢rst results from measuring the initial meanvelocities of matrix and analyte molecules in the IR-process showed no signi¢cant differences from UV^MALDI [ 101]. IR^MALDI has already found itsplace in the analysis of proteins immobilised on poly-mer membranes ( see Section 9) and will do so in theanalysis of very high mass compounds, for the reasonsdiscussed above.

9. UV^ and IR^MALDI-MS of proteinsseparated by gel electrophoresisfollowed by electroblotting

The mass analysis of single proteins of complexprotein mixtures, e.g. from whole cell lysates, can beperformed after separation of the mixture in gels(SDS-PAGE, 2D-PAGE). In the last two years, exper-imental protocols have been developed for obtainingthe mass information on intact protein masses, directlyfrom gels or from electroblots, by MALDI-MS. Thevalue of the intact-protein mass cannot be underesti-mated since it is speci¢c for the individual protein and,in many cases, determines the function of the proteinor, more speci¢cally, its processing state in the cell. Itmust not be forgotten that gel electrophoresis suffersfrom the principal limitation that it only permits thedetermination of electrophoretic mobilities, and notmolecular masses. While the idea of obtaining massinformation directly from the gel is more straightfor-ward, and does not lose any protein material, that ofperforming MALDI directly from electroblots has theadvantage that the mass spectrometry is not degradedby SDS molecules [ 102 ].

The mass information on the intact protein can beobtained directly from the gel, as has been followedintensively by Orgozalek Loo and co-workers[ 103,104 ]. It was found that isoelectric focusinggels show the most promise for obtaining high-qualityMALDI mass information directly from the gel. Thisis a consequence of the low tolerance of MALDItowards the ionic detergent SDS. In the experiments,SA and ACCA as matrices were applied to SDS gels orIEF gels, respectively. The gels were transferred to themass spectrometer and irradiated by UV lasers (337nm). Superior results were obtained from the IEF gels.

Isoelectric focusing strips ( IEF strips ) are used, inwhich the complex protein mixtures are separated bytheir pI values. MALDI-MS is taken as the seconddimension, and mass analysis taken directly from thegel serves as a detector for the large number of proteinshaving the same isoelectric point [ 105 ].

The strategy of performing MALDI-MS on intactproteins directly from electroblots was also followed[ 106,107 ]. It was found that the best method of sam-ple preparation is to incubate the membrane (PVDF)in aqueous matrix solution directly after the electro-blotting, while the blot is still wet from the electroblotprocedure [ 107 ]. By comparing the same electroblotpreparations in different wavelength ranges (266 or355 nm vs 2.94 Wm) it was found that superior resultswere obtained by IR^MALDI, in terms of reproduci-bility, single-shot intensities, and signal-to-noiseratio. Typically, only 10 single shots were suf¢cientto obtain a high quality mass spectrum [ 107,108 ]. Thereason for this is believed to lie in the much greaterpenetration depth of IR radiation into the membrane-matrix sample (compared to UV-radiation), whichresults in a much larger desorption depth, and there-fore a much larger number of protein molecules whichcan be ionised (see Section 8). It was shown that thelocal separation is preserved by using aqueous sol-vents for matrix incubation [ 109 ]. Sutton et al.[ 110 ] also applied the Er-YAG laser radiation to elec-troblotted proteins, using a similar preparation tech-nique, and came to the same main conclusion.

The UV^MALDI results reported by Patterson[ 111] and by Blais et al. [ 112 ], who also investigatedthe desorption / ionisation of proteins directly frommembranes, compare with the above described¢ndings that the spectra quality and reproducibilityare too poor for UV^MALDI to be used as a routinetechnique for immobilised proteins.

The latest results report the successful detection of2D-PAGE separated proteins of human plasma pro-teins. It was shown that the sensitivity can ^ at leastunder certain circumstances ^ be in the silver-stainingsensitivity range [ 113 ]. Since the mass information onthe intact protein can now be provided directly fromthe immobilising substrate, this technique wascoupled to on-membrane digestion followed by IR^MALDI mass analysis of the proteolytic fragmentsdirectly from the membrane. Details of this approach,and the results from database searches will be pub-lished [ 114 ].


TRAC 2481 8-9-98

Fig. 4. (A) Photomicrograph of an aggregate of human buccal mucosa (cheek) cells under UV £uorescent light. (B) Theselected ion image at m /z 7605, showing the distribution of IB-1 protein fragment bound to groups of aggregated cells. Theblack areas contain the highest concentration of protein, grey indicates moderate intensities, and the light grey area indicatesthe absence of signal from this protein. (C) Representative UV^MALDI spectrum obtained from cheek cells (100 laser shotsaveraged). Protein and peptides identi¢ed in the spectrum ( m /z ): (1 ) proline-rich protein-A (PRP-A), (M+H)� = 3805.7;(2) PRP-C, (M+H)� = 4374.3; (3) PRP-IB-4, (M+H)� = 5590.2; (4) PRP-IB-9, (M+H)� = 6023.8; (5) PRP-D,(M+H)� = 6947.8; (6 ) PRP-IB-1 fragment, (M+H)� = 7605; (7) PRP-IB-1, (M+H)� = 9585.1; (8) PRP-IB-6,(M+H)� = 11 503.8. The peak labelled `c' is a doubly charged ion of PRP-IB-9. From [ 116 ], with permission. (This imagecan be viewed in colour at http: / /www.elsevier.nl / locate / trac, click on Supplementary material. )


TRAC 2481 8-9-98

10. The way ahead: futuredevelopments of MALDI-MS with respectto molecular micro-probing

Following rapid development in the last ¢ve or sixyears, mass analysis by matrix-assisted laser desorp-tion / ionisation has become a routine technique inmany laboratories. Since MALDI-MS has already pro-vided answers to complex analytical questions, moresurprising results can be expected in the near future.One of the latest developments of MALDI relates tomolecular micro-probing.

10.1. Cell and tissue characterisation

Cell and tissue characterisation is interesting forclassi¢cation, phenotyping of mutants, investigationof cell-speci¢c compounds, or for elucidating patho-genic properties. For the characterisation of speci¢ccell compounds, single cell analysis is favourablesince less material is consumed (no pooling of mate-rial is needed) and characterisation is not falsi¢ed bymorphologically analogous cells. MALDI-MS is asensitive tool for identi¢cation of cell compounds,either directly from whole cells or from cell lysates,especially in combination with MS /MS techniques.Hsieh et al. interfaced microcolumn liquid chromatog-raphy ( micro-LC) with MALDI-MS for the separa-tion and identi¢cation of peptides that had beenreceived from single-cell lysates. The peptides areidenti¢ed by their elution positions in the reversed-phase chromatogram, their molecular masses in themass spectrum, and the generated PSD fragments[ 115 ]. The eluent is mixed off-line with the matrix(DHB and superDHB and, in the case of PSD analysis,the ACCA matrix ) and spotted directly onto theMALDI target. The feasibility of this approach wasdemonstrated with a giant neurone of the visceral gan-glion of the freshwater snail, Lymnaea stagnalis.

10.2. Screening of biological tissues

The screening of biological tissues, particularlywith respect to the spatial distribution of individualcompounds, provides a more detailed insight into thestructure of the tissue. It enables ^ after comparisonwith suitable libraries ^ the determination of patho-genic structures at the molecular level. Molecularimaging of biological samples was performed by Cap-rioli et al. in order to obtain `ion-surface maps' whichprovide information about the spatial arrangement of

different molecules in the analysed sample area [ 116 ].The overall image of a given tissue is accomplished byscanning the area of interest in an ordered array ofmass spectra, with each mass spectrum containinginformation about biological compounds accordingto their speci¢c mass values. The spatial distributionof one of these compounds can be envisioned by plot-ting its mass in two dimensions along the x and y axesof the tissue. The ion-signal intensity of the (M+H)�value is shown in the plot as the third dimension.

Fig. 4 shows the analysis of human buccal mucosacells after they have been spread onto a stainless steeltarget, stained with 0.2% methylene blue for visual-isation with UV £uorescent light, then dried and cov-ered with matrix. Identi¢cation of the data set can bemade with the help of proteolytic digests and data-bases.

10.3. Classi¢cation and detection of bacteria

The classi¢cation and detection of bacteria isanother interesting ¢eld of direct cell analysis. Thegrowth of bacteria for speci¢cation of the strain isrelatively time-consuming and inef¢cient for the treat-ment of patients, so new methods which would allowthe rapid characterisation of the strain would be veryhelpful. The fast analysis of infected tissue could resultin the treatment of a patient with selective antibiotics( instead of broad-spectrum antibiotics ), which wouldbe very ef¢cient in combating the spreading of resist-ant organisms. The approach of Krishnamurthy andRoss [ 117 ] and of Claydon et al. [ 118 ] is based onthe detection of the genus, species and strain-speci¢cbiomarkers such as soluble cell-surface components ormolecules from osmotic cell lysis. It has beenobserved that the presence of these biomarkers,which are determined empirically, is independent ofthe growth conditions, the pH value of suspensionbuffers, or changes in the preparation protocols;also, the results were reproducible. Even bacteria invegetative or sporulated forms showed identical bio-markers, although distinct ions were observed whichcould enable the determination of the growth condi-tions, and even any sources of bacterial contamina-tion.

The characterisation of bacteria with speci¢c bio-markers, in combination with computer-stored libra-ries, is suited for ¢ngerprint screening of potentiallycontaminated material. Direct tissue examinationwould be especially bene¢cial in ¢ghting infectionsrapidly.


TRAC 2481 8-9-98

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Julia Gross is a Ph.D. student in Biochemistry in the groupof Franz Hillenkamp at the University of Muënster,Germany. Her main ¢eld of interest is the mass analysisof oligonucleotides. She is working on the elucidation ofMALDI-induced fragmentations and the development ofsequencing procedures. Julia Gross received herDiploma in Biochemistry from the University of Bochum,Germany, in 1996 for a work in the ¢eld of proteinchemistry.

Kerstin Strupat is a research scientist in the group ofFranz Hillenkamp. She got her Diploma (1991) andPh.D. (1995) in Physics, both from the University ofMuënster. Her ¢elds of interest are the understanding ofmatrix^analyte interaction in MALDI samples, the massanalysis of proteins electroblotted onto polymermembranes by MALDI, and the analysis of non-covalentcomplexes by ESI^MS and MALDI^MS. After her Ph.D.she spent one year in the Protein Chemistry group ofFriedrich Lottspeich at the Max-Planck-Institute forBiochemistry in Munich.

Laser-induced £uorescence detection inmicrocolumn separationsLi Tao, Robert T. Kennedy*Chemistry Department, University of Florida, P.O. Box 117200, Gainesville, FL 32611-7200, USA

Laser-induced £uorescence (LIF) has beenused extensively in capillary separationsdue to its high sensitivity and selectivity.This article highlights the history and recentadvances and applications of LIF detection incapillary electrophoresis and capillary chro-matography. z1998 Elsevier Science B.V.All rights reserved.

Keywords: Laser-induced £uorescence;Fluorescence detection, laser-induced

1. Introduction

Perhaps the fastest growing area of chemical sepa-rations research is in the development of microscale

techniques such as capillary liquid chromatography(LC) and capillary electrophoresis (CE). The tremen-dous interest in microcolumns results from severalwell-known advantages over conventional scale meth-ods including higher resolution, compatibility withsmaller samples, and consumption of less mobilephase or buffer. All of these advantages are achievedby miniaturizing the separation path to columns withinner diameters of 1^100 Wm. While the potential ofcapillary scale separations is extremely high, realiza-tion of this potential for `real-world' analytical meas-urements is dependent upon developing suitableinstrumentation and methodology. Perhaps the mostcrucial and dif¢cult problem to solve is that of detec-tion. To avoid extracolumn band broadening, injectionvolumes are typically 0.1^10 nl in capillary LC andCE. For an analyte present at 1 nM concentration, a0.1-nl injection volume requires detection of 100 zmolof analyte. Quanti¢cation of this concentration would

0165-9936/98/$ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 5 - 9 9 3 6 ( 9 8 ) 0 0 0 5 6 - 9

*Corresponding author.


Matrix-assisted laser desorption/ionisation–mass spectrometry applied to biological macromolecules

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