Introduction Ion sources & sample introduction Mass ... · PDF fileMass SpectrometryMass...

Mass SpectrometryMass SpectrometryMass Spectrometry

-- IntroductionIntroduction

-- Ion sources & sample introductionIon sources & sample introduction

-- Mass analyzersMass analyzers

-- Basics of Basics of biomoleculebiomolecule MSMS

-- ApplicationsApplications

Adapted from Adapted from ““Mass Spectrometry in BiotechnologyMass Spectrometry in Biotechnology””

Gary Gary SiuzdakSiuzdak, Academic Press 1996, Academic Press 1996

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Mass SpectrometryMass SpectrometryMass SpectrometryIntroductionIntroduction-- In the past, mass spectrometry was confined to the realm of smalIn the past, mass spectrometry was confined to the realm of small molecules; l molecules;

large molecules did not survive the desorption and ionization prlarge molecules did not survive the desorption and ionization process intact.ocess intact.

-- MatrixMatrix--assisted laser desorption/ionization (MALDI).assisted laser desorption/ionization (MALDI).

-- Electrospray ionization (ESI)Electrospray ionization (ESI)

-- Fast atom/ion bombardment (FAB)Fast atom/ion bombardment (FAB)

-- The analysis of proteins, peptides, carbohydrates, oligonucleotiThe analysis of proteins, peptides, carbohydrates, oligonucleotides, natural des, natural

products and drug metabolites.products and drug metabolites.

-- Detection capabilities ranging from the picomole to the femtomolDetection capabilities ranging from the picomole to the femtomole level.e level.

-- Molecular weight accuracy on the order of Molecular weight accuracy on the order of 0.010.01%.%.

-- The utilities of MS include molecular weight characterization, nThe utilities of MS include molecular weight characterization, noncovalent oncovalent

interactions, protein and peptide sequencing, DNA sequencing, printeractions, protein and peptide sequencing, DNA sequencing, protein otein

folding, in vitro drug analysis, and drug discovery. folding, in vitro drug analysis, and drug discovery.

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Mass SpectrometryMass SpectrometryMass SpectrometryIntroductionIntroduction

Light source

Light detector

Prism

m / z

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Mass SpectrometryMass SpectrometryMass Spectrometry-- MatrixMatrix--assisted laser desorption/ionizationassisted laser desorption/ionization

-- A nonvolatile solid as the matrix.A nonvolatile solid as the matrix.

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Mass SpectrometryMass SpectrometryMass Spectrometry-- MatrixMatrix--assisted laser desorption/ionizationassisted laser desorption/ionization

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Mass SpectrometryMass SpectrometryMass Spectrometry-- Electrospray ionizationElectrospray ionization

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z1

z1-1

myoglobinz1 = 11

M = 16,951

z1 x 1542 = M + z1(z1-1) x 1696 = M + (z1-1)

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Mass SpectrometryMass SpectrometryMass SpectrometryMass analyzersMass analyzers

-- QuadrupoleQuadrupole

-- TimeTime--ofof--flight & timeflight & time--ofof--flight reflectronflight reflectron

-- Ion trapIon trap

-- MagneticMagnetic

-- DoubleDouble--focusing magnetic sectorfocusing magnetic sector

-- FTFT--IR (ion cyclotronIR (ion cyclotron9

Mass SpectrometryMass SpectrometryMass SpectrometryMass analyzersMass analyzers

Resolution is the ability of a mass spectrometer to distinguish Resolution is the ability of a mass spectrometer to distinguish

between ions of different massbetween ions of different mass--toto--charge ratios. Therefore, charge ratios. Therefore,

greater resolution corresponds directly to the increased abilitygreater resolution corresponds directly to the increased ability to to

differentiate ions.differentiate ions.

Resolution = M / Resolution = M / ∆∆M = MM = M11 / (M/ (M11 –– MM22))

∆∆M = full width at half maximum (FWHM)M = full width at half maximum (FWHM)

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Mass SpectrometryMass SpectrometryMass Spectrometry-- Quadruple analyzerQuadruple analyzer

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Mass SpectrometryMass SpectrometryMass Spectrometry-- TimeTime--of of ––flight analyzer & timeflight analyzer & time--ofof--flight reflectronflight reflectron

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Mass SpectrometryMass SpectrometryMass Spectrometry-- Tandem mass spectrometryTandem mass spectrometry

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Mass SpectrometryMass SpectrometryMass Spectrometry-- Tandem mass spectrometryTandem mass spectrometry

ABCABCDEFDEF

mass ABCmass ABConlyonly

AB, BCAB, BCABCABC

scan allscan allmassesmasses

ABCABCDECDEC

scan all scan all massesmasses

AB, DEAB, DEC, etc.C, etc.

mass Cmass Conlyonly

ABCABCDBFDBF

scan all scan all massesmasses

AC, DFAC, DFAB, etc.AB, etc.

mass mass minus Bminus B

ABCABCDEFDEF

mass ABCmass ABConlyonly

AB, BC AB, BC ABC*ABC*

mass BCmass BConlyonly

BCAB

ABC

DECABC

BC

DFAC

子離子掃瞄子離子掃瞄Daughter ScanDaughter Scan

母離子掃瞄母離子掃瞄Parent ScanParent Scan

中性丟失掃瞄中性丟失掃瞄Parent ScanParent Scan

選擇反應偵測選擇反應偵測SRMSRM

離子源離子源質譜儀質譜儀--II 質譜儀質譜儀--IIII解離室解離室質譜圖質譜圖

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Mass SpectrometryMass SpectrometryMass SpectrometryBasics of Basics of biomoleculesbiomolecules mass spectrometrymass spectrometry-- 1.1. Salt content should be minimized.Salt content should be minimized.

2.2. Matrix selection/preparation.Matrix selection/preparation.3.3. Care should be taken to maintain high purity.Care should be taken to maintain high purity.4.4. Sample solubility in the solvent or matrix is crucial.Sample solubility in the solvent or matrix is crucial.5.5. Is there enough amount of sample?Is there enough amount of sample?6.6. The functional groups help to determine how to analyze.The functional groups help to determine how to analyze.7.7. Analyze soon after synthesis and/or purification. Analyze soon after synthesis and/or purification.

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Mass SpectrometryMass SpectrometryMass SpectrometryBasics of Basics of biomoleculesbiomolecules mass spectrometrymass spectrometry

Solubility: 14-kDa protein electrospray mass analyzed

methanol

33% methanol33% acetonitrile33% water

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Mass SpectrometryMass SpectrometryMass SpectrometryBasics of Basics of biomoleculesbiomolecules mass spectrometrymass spectrometry

MALDI matrix

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Mass SpectrometryMass SpectrometryMass SpectrometryApplicationsApplications

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Mass SpectrometryMass SpectrometryMass SpectrometryApplicationsApplications

Protein folding

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} TagIdent

Overview of protein identificationOverview of protein identification

Adapted from Micromass and “Proteome Research: New Frontiers in Functional Genomics”

-- Primary attributes & Secondary attributes.Primary attributes & Secondary attributes.-- Most attributes relate directly or indirectly to Most attributes relate directly or indirectly to

a proteina protein’’s sequence, however they vary in s sequence, however they vary in the way that they are generated and the the way that they are generated and the protein property they represent.protein property they represent.

-- Crossed references.Crossed references.21

Mass SpectrometryMass SpectrometryMass SpectrometryESIESI-- Tandem MSTandem MS MALDIMALDI--TOF MSTOF MS

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Mass SpectrometryMass SpectrometryMass SpectrometrySample Preparation for Mass Spectrometric AnalysisSample Preparation for Mass Spectrometric Analysis

-- Pure proteins or protein spots from 1Pure proteins or protein spots from 1--D or 2D or 2--D gel electrophoresisD gel electrophoresis

-- Protein digestion with proteases to yield fragments that are mosProtein digestion with proteases to yield fragments that are most compatible t compatible

with MS analysis, 6 with MS analysis, 6 -- 20 amino acids20 amino acids

(i.e. Trypsin: a 50kDa to 30 (i.e. Trypsin: a 50kDa to 30 tryptictryptic

peptides).peptides).

-- NonNon--specific protease and specific protease and CNBrCNBr..

-- Extraction of peptides from gel spots.Extraction of peptides from gel spots.

-- Exact masses of the resulting peptides.Exact masses of the resulting peptides.

-- Fragmentation of peptide ions in MS analysis.Fragmentation of peptide ions in MS analysis.

-- 5 5 -- 10 10 fmolfmol level for peptide mapping.level for peptide mapping.

-- 50 50 -- 100 100 fmolfmol level for peptide sequencing.level for peptide sequencing.23

Mass SpectrometryMass SpectrometryMass SpectrometryPeptide mass fingerprinting (PMF) or mappingPeptide mass fingerprinting (PMF) or mapping

*

***

**

*

Trypsin

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Mass SpectrometryMass SpectrometryMass SpectrometryPeptide mass fingerprinting (PMF) or mappingPeptide mass fingerprinting (PMF) or mapping

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Mass SpectrometryMass SpectrometryMass SpectrometryPeptide mass fingerprinting (PMF) or mapping using MASCOTPeptide mass fingerprinting (PMF) or mapping using MASCOT

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Mass SpectrometryMass SpectrometryMass SpectrometryPeptide fragmentation to generate partial sequencePeptide fragmentation to generate partial sequence

Sequencing

Fragmentation

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Mass SpectrometryMass SpectrometryMass SpectrometrySelection of charged peptidesSelection of charged peptides

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Mass SpectrometryMass SpectrometryMass SpectrometryTransformation & deTransformation & de--isotopingisotoping of raw MSof raw MS--MS data.MS data.

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Mass SpectrometryMass SpectrometryMass SpectrometrySequencing results from MSSequencing results from MS--MS of m/z 841.5 (M+3H)MS of m/z 841.5 (M+3H)3+3+

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Mass SpectrometryMass SpectrometryMass SpectrometrySequence readingSequence reading

L = I, Q/K = G+A, W = G+E = A+D31

Mass SpectrometryMass SpectrometryMass SpectrometrySequencing results from MSSequencing results from MS--MS of m/z 841.5 (M+3H)MS of m/z 841.5 (M+3H)3+3+

β-Galactosidase

Q (89%) versus G (9%) A (4%) at position 3W (35%) versus G (65%) E (65%) at position 18G (80%) E (80%) versus A (19%) D (19%) at position 21

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Mass SpectrometryMass SpectrometryMass SpectrometryPeptide sequencing using MASCOTPeptide sequencing using MASCOT

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Mass SpectrometryMass SpectrometryMass SpectrometryPeptide sequencing using MASCOTPeptide sequencing using MASCOT

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Mass SpectrometryMass SpectrometryMass SpectrometryProtein identification by peptide sequencingProtein identification by peptide sequencing

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Mass SpectrometryMass SpectrometryMass SpectrometryPartial sequencing plus MALDIPartial sequencing plus MALDI--TOFTOFPeptide sequencing using Peptide sequencing using

chemical modificationschemical modifications

lysine to homoarginine

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Amino acid residue modificationsAmino acid residue modifications

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Chemical modificationsChemical modifications

Mass SpectrometryMass SpectrometryMass SpectrometryPostPost--translational modificationstranslational modifications

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Mass SpectrometryMass SpectrometryMass SpectrometryQuantitative analysis using isotopeQuantitative analysis using isotope--coded affinity tags (ICAT)coded affinity tags (ICAT)

Nature biotechnology (1999) 17:994-999

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Introduction Ion sources & sample introduction Mass ... · PDF fileMass SpectrometryMass...

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