Oct 2011 SDMBT

Lecture 9Mass Spectrometry

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Objectives

Describe the principle of Mass Spectrometry

In particular, the ionisation methods(a) MALDI(b) ESI

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General workflow for proteomic analysis

SampleSample preparation

Protein mixture Sample separation and visualisation

Comparative analysis

DigestionPeptides

Mass spectrometry

MS dataDatabase search

Protein identification

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Protein Identification

MALDI-TOF

Molecular weight of tryptic peptides

ESI-MS

Molecular weight of protein

MS/MS

Molecular weight of peptide fragments

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Mass spectrometry workflowTypical process

(Medical College of Georgia)

ionisation

acceleration

sorting

detection

Mass-to-charge ratio

(m/z)

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The need for ionisation

Analyte has to be converted into gas-phase ions – only charged ions can be detected by MS

Movement of gas-phase ions can be precisely controlled by electromagnetic fields

Difficulty of generating gas-phase ions results in complex instruments and high costs – need high vacuum

Gas phase ions can be generated by

e.g. an electron beam (electron ionisation)

collision with inert gas molecules (MS-MS)

a laser beam (MALDI-TOF)

a charged nozzle (ESI-MS)

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A plot mass of ions (m/z) (x-axis) versus the intensity of the signal (roughly corresponding to the number of ions) (y-axis)

Mass spectrum of water – ionised by electron ionisationNotice that the water molecule fragments upon ionisation

Mass Spectrum

H2O+

HO+

O+

H+

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HCHO+

CHO+

CO+

HCHO2+

HCH+

CH+

C+

Notice: the horizontal axis is mass/charge ratio of ions (m/z)

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The need for ionisation

Proteins are large macromolecules – electron ionisation works only for volatile compounds – also electron ionisation is a ‘hard’ ionisation technique – molecule fragments into smaller ions

Need to find an efficient method to convert proteins from liquid phase to gas-phase ions

Soft ionisation methods like electrospray ionisation (ESI) and matrix-assisted laser-desorption ionisation (MALDI)

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Soft ionisationIonise peptides into gas-phase ions

Too much energy (hard ionisation) breaks peptides into smaller fragments

(Prof. Jose-Luis Jimenez)

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Maldi TOF/TOF mass spectroscopic spectre for UCH-L3 digested Ecotin-Ubiquitin-FLS in the m/z range 799–4013 . The peaks

at 2683 and 1342 represent the single and double protonation states of the FLS peptide, respectively.

MALDI-TOF mass spectrumPeptide does not fragment

Soft ionisation

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MALDI

Peptides are mixed with a matrix, which crystallise on a metal plate

Peptides protonated by matrix and solvent

Pulses of laser transfer energy to matrix

Matrix vaporises with peptide samples

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MALDI

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MALDI plate

(Bruker Daltonics)

(University of Pittsburgh BRSF)

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MatrixContains ring structures to absorb energy from UV laser

Contains acid group to protonate peptides

OH

OH

HOOC

2,5-dihydroxybenzoic acid

OH

COOH

CN

-cyano-4-hydroxycinnamic acid

Matrix solution – DHB or CHCA dissolved in acetonitrile and trifluoroacetic acid

Sinapinic acid

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Characteristics of MALDI

Efficient protonation of peptides

Ions generated in discrete packets due to laser pulses

Combined with time-of-flight (TOF) mass analysis to give very high sensitivity

More tolerant than ESI to contaminants (urea, SDS), thus HPLC separation is not required

                                                                     

      

MALDI-TOF of mixture of cytochrome, ubiquitin, myoglobin(without PSD) – note single peaks (except cytochrome) – cantell Mw of 3 proteins in one spectrum

Mw of myoglobin17000

Mw of cytochrome C 12000

Mw of ubiqutin 8500

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Trypsin digested peptides

(ExPasy PeptideCutter)

Arginine or Lysine Proline

From last lecture: one spot in 2D-gel represents one protein – this proteinis digested with trypsin into smaller peptides.The peptide mixture is spotted on the MALDI plate

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King’s College London

(Pierce)

Major peaks at:

646 742 748

780 830 2186

Represent expected sizes of tryptic peptides

Soft ionisation ensures no further fragmentation of peptides

MALDI spectrum of Tryptic digest of β-casein

Each peak represents one peptidedigested by trypsin from casein

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Tryptic digest of β-casein

King’s College London

(Pierce)

Most abundant peak base peak, abundance set to 100%

MS is seldom quantitative, only tells relative abundance

Ideally every peptide is ionised to same extent 100%

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Electrospray Ionisation (ESI)Peptides in acidic solution are pushed out of a fine capillary

A high positive charge at the capillary results in the formation of a Taylor cone

Peptides protonated in charged droplets and ionised

Gas-phase ions repelled from capillary into instrument

Nitrogen aids in evaporation of solvent from charged droplets

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Electrospray Ionisation (ESI)

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High voltage results in accumulation of positive charges on droplets

Increased charge density results in instability of droplets

Droplets break down successively into smaller sizes

Electrospray Ionisation (ESI)

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Characteristics of ESI

Acidic conditions result in protonation of all basic sites in peptides

Basic side groups of lysine, arginine, histidine

Produces peptide ions that are multiply protonated

Advantageous for peptides digested by trypsin as doubly charged peptides are formed

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Characteristics of ESI

Efficient ionisation process results in sensitivity of ESI experiments

General compatibility of ESI with reversed-phase high performance liquid chromatography (RP-HPLC) LC/MS-MS

ESI-MS - often see doubly, triply, multiply charged ions

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ESI of myoglobin

Mw=1413.8x12-12 = 16953.6

Mw – need to know how to interpret by assigning charges

Compare with slide 17More complicated Different scale on horizontal axis

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ESI of cytochrome C

Compare with slide 17More complicated Different scale on horizontal axis

ESI of ubiquitin

5+

Mw = 8484.5

6+

7+

Compare with slide 17More complicated Different scale on horizontal axis

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MALDI-TOF – 3 peaksEach of each protein

ESI – complicatedif sample was a mixture – 3 superimposedspectra

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MALDI-TOF ESI

Sample Solution but ends upembedded in crystalline matrix

Sample tolerant to salts

Singly charged ions

Adduct-formation not so

Common.

Appearance – one peak

Solutioneg can come straight from HPLC

Results can be affected by salts eg phosphates

Multiply-charged ions

Adducts with salts common

May be difficult to interpret spectra

Appearance – many peaks

due to multiple charges

Comparison of ionisation methods

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MALDI-TOF ESI

Ionisation Laser Charged spray nozzle

Mass analyser TOF Quad

Fragmentation is made possible by

PSD (Post-Source decay)

“Pseudo-MS-MS”

MS-MS or Tandem MS

Ions fragmented by collision with inert gas

Use Mainly peptides Peptides and proteins

Comparison of ionisation methods

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Vacuum SystemVacuum System

Sample Sample InletInlet DetectorDetector

Data Data SystemSystem

Mass Mass AnalyserAnalyser

Ionisation Ionisation MethodMethod

Components of a mass spectrometer

Atmosphere

(adapted from Thermo Finnigan)

MALDI

ESI

TOF

Quadrupole

Ion trap

Combination

ionisation acceleration detection

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The need for a mass analyser

Gas-phase ions has to be filtered/arranged in order to allow selection of specific ions for further analysis.

Movement of gas-phase ions precisely controlled by use of electromagnetic fields in a mass analyser

Main types of mass analyser-TOF (time-of-flight analyser)

-Quadrupole analyser

-Fourier-Transform Ion Cyclotron Resonance

-Orbitrap

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TOF

Time-of-flight (TOF) analyser is the simplest mass analyser

Peptide ions accelerated into flight-tube, and maintains a velocity due its given kinetic energy

TOF analyzer requires that ions are introduced in a pulse well-suited for MALDI

Resolution increases with length of TOF tube

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Linear TOF

(John Lennon, University of Washington)

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MALDI- TOF with reflectron

Reflectron - This turns ions around in an electric field, sending

them towards the detector – improves mass resolution

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Quadrupole

(NASA)

Made up of 4 parallel gold barsComplex electromagnetic fieldset up which allows only ions of aa set mass/charge ratio through

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Flight path of an ion through a quadrupole

Correct m/z ratio Larger/smaller m/z ratio

Ions that spiral out of control crash into the rods or casing

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Fourier Transform Ion cyclotron resonance (FT-ICR)

See this websitehttp://www.chm.bris.ac.uk/ms/theory/fticr-massspec.html

Magnetic fields applied to trapping plates constrain the ions to move around in a circlein between the plates. The circular motion induces an alternating current.The frequency of the AC is related to the m/z ratio.

Main advantage of FT-ICRis very high mass resolution

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Orbitrap

Image from thermo.com

Similar to FT-ICRElectric field applied to trapping plates constrain the ions to move around in a circlein between the plates. The circular motion induces an alternating current.The frequency of the AC is related to the m/z ratio.

Inner electrode

Outer electrode

Main advantage of Orbitrapis very high mass resolution

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Tandem MS (MS/MS)

MALDI and ESI are soft ionisation techniques – peptides or proteinsdo not fragment – useful for molecular weight determination

However sometimes fragmentation is useful because it give useful informationabout the amino acid sequence of peptides (next lecture)

Fragmentation can achieved by a number of ways

-Post source decay (PSD)-Collision induced dissociation (CID)-Infrared multiphoton dissociation-Electron capture dissociation (ECD)-Electron transfer dissociation (ETD)

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Post-Source Decay (PSD)

- Use higher laser power or introduce inert gas to collide with ions so that ions fragments between source and TOF

- Method of fragmenting intact peptides - get amino acid sequence information (see next lecture)

- Electronics to select ions to go into the TOF

http://abrf.org/ABRFNews/1995/December1995/dec95maldi.html

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(K. Yoshinari, Rapid Commun. Mass Spectrom. 14, 215-223, 2000)

Ion trap

(NASA)

All other dissociation techniques involve the use of an ion trapIon trap essentially a quadrupole where the magnetic field is set such that a particular ion is trapped in the space between the electrodes

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Ion trap

(March, JMS Vol. 32, 351-369, 1997 )

-Collision induced dissociation (CID) – inert gas (e.g. Xe, Ar, N2 or He) is introduced into trap, collisions will cause peptides to fragment usually the C-N peptide bond to produce b and y ions (see later)-Infrared multiphoton dissociation (IRMPD) – IR laser is fired into ions to excite the vibrations of peptides. -Electron capture dissociation (ECD) – electron beam is fired into ions to produce c and z ions-Electron transfer dissociation (ETD) – singly charged anion, fluoranthracene is introduced. An electron is transferred to peptide. Results in c and z fragments

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Hybrid mass analysers

Basically a combination of two types of mass analysers or many of the same type

Each analyser performs a different function

Used to perform tandem mass spectrometry, using collision induced dissociation (CID)

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Tandem quadrupoles (QQQ/TSQ)

Video

Quantitative Chemical Analysis

Select specific parent ion

CID with inert gas

Scanning of all m/z of daughter ions

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Tandem mass spectrometry

A) MS spectrum of HSP27. The peptide whose MS/MS spectrum is shown in panel B is indicated. B) MS/MS spectrum of the peptide ion m/z 1163 obtained in CID mode.Perroud et al. Molecular Cancer 2006 5:64