醫學新知導論醫學新知導論

Mass Spectrometry in BiotechnMass Spectrometry in Biotechn

ology/Proteomicsology/Proteomics

Yi-Ting Chen ( 陳怡婷 ), Ph.D.

Molecular Medicine Research Center

Chang Gung University

Dec. 4, 2008

Outline• Introduction of mass spectrometry

– principle– Instrumentation

• Proteomics– Introduction– Mass spectrometry in proteomics– Protein identification– Quantitative proteomics– Post-translational modification

• Application of mass spectrometry in biotechnology– Food science– Environment chemistry– Drug analysis

The Five Mass Spectrometry Nobel Prize Pioneers

WHAT IS A “MASS SPECTROMETER ”...?

MS

The black box problem…...

ESI MALDI LC/MS LC-MSMSESI

ESI Ion trap TOF QTOFquad.

ESI CRIMS EI APCIFAB

FT-ICR

qQTOF

SELDI

…many black boxes !

“A mass spectrometer measures the molecular weight….”

“...A MS analysis gives

the mass-to-charge ratio (m/z) of ions…in gas phase”.

Data Processing

Ion Source

Analyzer

ion separation

vacuum

Detector

Pumping system

Sample introduction

Introduction(solid, liquid, gas)

Separation technique (HPLC, CE, GC)

ESI, nano ESI, MALDI, FAB, EI, CI, APCI, SIMS…

TOF, quadrupole, Ion trap, FT, magnetic sectorQQQ, Q/TOF, Q/IT…

h

Nd:YAG Laser355 nm

Sample ions, MH+

Ground Grid

Sampleplate

High voltage

Sample & matrix

To Mass Analyzer

Matrix Assisted Laser Desorption Matrix Assisted Laser Desorption Ionization (MALDI)Ionization (MALDI)

HPLC - MS

• Ion Source ( 離子源 )– 大氣壓下游離法 (Atmosp

heric Pressure Ionization, API)

• 電灑法 – Electro-spray, ESI

• 大氣壓化學游離法– Atmospheric Pressure

Chemical Ionization, APCI

Analyte Polarity

ESI

APcI

Molecular Weight

1000

1000000

EI600

Electro-spray, ESI ( 電灑法 )

Desolvation

泰勒錐Taylor cone Cone

voltage

pumping

HPLC - MS

• Electro-spray, ESI ( 電灑法 )

Capillary ~3 kV

As droplets evaporate, the electric field increases and ions move towards the surface.

Ions evaporate from the surface

Solvent evaporationCoulombic explosion

0

20

40

60

80

100

%Int.

5000 10000 15000 20000Mass/Charge

Data: 1015pl30001.O7 15 Oct 2002 16:22 Cal: 16 Oct 2002 8:12

8480.6

5653.4

16953.04239.4

MALDI-TOF spectra of apomyoglobin

INSTRUMENT: Kratos Axima-CFR

Sample: 1 pmole apomyoglobin (horse skeletal muscle)

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

+101695.9

+111541.8

+121413.5

+131304.8

+141211.7

+15

1131.0

+16

1060.4

+17

998.1

+18

942.8

+19893.2

+20848.6

+21808.2

+22771.5

+23738.0+24

707.4+25

679.1

+91884.5

ACTUAL SPECTRUM

+91884.3

16000 16400 16800 17200 17600 18000 18400

mass

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

16951.5

AFTER DECONVOLUTION

ESI-ion trap spectra of apomyoglobin

INSTRUMENT: Thermoquest LCQ-classic

Sample: 1 pmole apomyoglobin (horse skeletal muscle)

HPLC -MS• Atmospheric Pressure Chemical Ionization, APCI ( 大

氣壓化學游離法 )

• http://www.chm.bris.ac.uk/ms/theory/apci-ionisation.html

HPLC- MS

TechniqueFlow Rate

(ml/min)M.W Range

Species

Produced

ESI 0.001-0.3 ＜ 2000000(M+H)+

(M-H)-

(M+nH)n+

APCI 0.2-2.0 ＜ 1000(M+H)+

(M-H)-

ESI and APCI differ in…• How ions are generated

• ESI - solution phase ionization• APCI - gas phase ionization

• Analyte compatibility• ESI - polar compounds and large biomolecules• APCI - less polar, smaller compounds (relative to th

ose ionized by ESI) that have some volatility

• Flow rate compatibility• ESI - 0.001 to 1 mL/min• APCI - 0.2 to 2 mL/min

HPLC -MS• Effect of Flow Rate

Flow rate (ml/min) (50/50 ACN/H20)

0 0.5 1.0 1.5 2.0

50

100

Rel

ativ

e in

tens

ity

APCI

ESI

HPLC -MS• Quadrupole

To Detector

-+

-+

From Source

X

Y

Volta

ge o

n Ro

ds-v

e

0

+ve

RF Cycle

X

Y

MALDI Time-of-flight (TOF) MSMALDI Time-of-flight (TOF) MS

source

TOF

Mass (m/z)

inte

ns

ity

Laser

The ions enter the flight tube with the lighter ionstravelling faster than the heavier ions

                                                                            

m/z is mass-to-charge ratio of the ionE is the extraction pulse potentials is the length of flight tube over which E is appliedd is the length of field free drift zonet is the measured time-of-flight of the ion

Ion Trap

Fourier-transform Ion Cyclotron Resonance (FT-ICR)

High resolution and accuracy

Single MS Analyzer

mass scanning mode

m1m3m4 m2

m3

m1

m4

m2

single mass transmission mode

m2 m2 m2 m2m3

m1

m4

m2

m/z

HPLC-MS/MS Triple Quadrupole

1. Samples from the liquid 1. Samples from the liquid introduction system enter the introduction system enter the ionisation source at atmospheric ionisation source at atmospheric pressurepressure

1. Samples from the liquid 1. Samples from the liquid introduction system enter the introduction system enter the ionisation source at atmospheric ionisation source at atmospheric pressurepressure

2. Ions are transferred 2. Ions are transferred to the analysers to the analysers through a hexapole through a hexapole lenslens

2. Ions are transferred 2. Ions are transferred to the analysers to the analysers through a hexapole through a hexapole lenslens

3. The ions are filtered in 3. The ions are filtered in MS1 according to their mass MS1 according to their mass to charge ratio (m/z)to charge ratio (m/z)

3. The ions are filtered in 3. The ions are filtered in MS1 according to their mass MS1 according to their mass to charge ratio (m/z)to charge ratio (m/z)

4. The mass separated 4. The mass separated ions undergo CID in the ions undergo CID in the hexapole collision cellhexapole collision cell

4. The mass separated 4. The mass separated ions undergo CID in the ions undergo CID in the hexapole collision cellhexapole collision cell

5. The fragment ions are 5. The fragment ions are filtered in MS2 filtered in MS2 according to their mass according to their mass to charge ratioto charge ratio

5. The fragment ions are 5. The fragment ions are filtered in MS2 filtered in MS2 according to their mass according to their mass to charge ratioto charge ratio

6. The ions are detected by 6. The ions are detected by an off-axis photomultiplier an off-axis photomultiplier detector.detector.

6. The ions are detected by 6. The ions are detected by an off-axis photomultiplier an off-axis photomultiplier detector.detector.

MS/MS Scan Functions

mass scan modesingle mass transmission

m2 m2 m2 m2m3

m1

m4

m2

Collision Chamber (gas)Collision Chamber (gas)

++

+

+

+

+

N2

+ + + ++

Q1 Q3Product Ion Scan (PI) Fix ScanMultiple Reaction Mode (MRM) Fix FixPrecursor Ion Scan (PS) Scan FixNeutral Loss Scan (NL) Scan Scan

Product Ion Scanning

mass scan modesingle mass transmission

m2m2 m2m2 m2m2 m2m2m3m3

m1m1

m4m4m2m2

Collision Chamber (gas)

++++

++++

++

++

NN22

++++ ++ ++

Parked on Precursor ion

Parked on a product Ion CAD

Q1 Q3Q2

Most sensitive scan type for detection of known components

- Q1 is set on the parent ion m/z (usually multiply charged for peptides)- ions are fragmented in Q2 collision cell - Q3 is set on the diagnostic fragment m/z

Fundamental to Absolute Quantitation is the Triple Quad Scan termed Multiple Reaction Monitoring (MRM)

Precursor Ion Scanning

Scan Precursors Select ProductCAD

Q1 Q3Q2

Only ions passed through Q1 that produce the PTM-specific fragment mass (e.g. 79 for phosphorylation or 204 for glycosylation) will produce signal at the detector

http://www.sciencemag.org/cgi/content/full/291/5507/1221/F1

• Proteomics– Introduction– Mass spectrometry in proteomics– Protein identification– Quantitative proteomics– Post-translational modification

• Proteome : In 1993, the term “Proteome”, by Marc Wilkins and Keith Williams, was referred to the systematic identification of the entire protein population expressed by a genome or by a cell or tissue type.

• Proteomics : The subject of proteomic analysis of the proteome (PROTEin complement expressed by a genOME or by a cell or tissue type).

(Wilkins et al., 1995 Biotechnology and Genetic Engineering Reviews 13, 19-50.)

Definition of ProteomicsDefinition of Proteomics• Yates defined proteomics as the scientific discipline of characterizin

g and analyzing the proteins, protein interactions, and protein modifications of an organism.

• Gygi and Aebersold defined proteomics as the ability to systematically identify every protein expressed in a cell or tissue tissue as well as to determine the salient properties of each protein, i.e., abundance, state of modification, involvement in multiprotein complexes, etc.

• Wagner defined the proteome is the entire profile of all the proteins expressed by a cell or a tissue under strictly defined conditions at a given time.

-proteomics aims to: -separate identity and characterize proteins on a large scale -define levels of proteins in cells / tissues and how these change -investigate protein complexes -elucidate protein functions, pathways, and interrelationships

蛋白體的發展里程碑蛋白體的發展里程碑

Why analyze gene expression at the protein level?

Y

YRNA Proteins Modified

ProteinsDNA Biological

Function

Transcription Translation Post-TranslationModification

Genome Transcriptome Proteome

<30,000 Genes > 1,000,000 Proteinsx 5 to 50

functionallinks per protein

Why the study of proteins is so challenging?

A polypeptide can fold to generate a particular three-dimensional structure specified by its amino acid sequence.

The structural description of proteins is described in terms of four levels of organization.

MS-based ProteomicsMS-based Proteomics

Qualitative

Protein identificationPeptide Mass Fingerprint (PMF)

MS/MS ion searching

De novo Sequencing

Post Translation Modification (PTM)

Quantitative

Protein quantitation

ICAT

iTRAQ

SILAC

O16/O18

Non-labelling tech.

MRM

The general Property Differences The general Property Differences between DNA and Proteinbetween DNA and Protein

mRNA level expressed protein level

Kind of Material DNA PROTEINResidues 4 20

Size define large

Concentration even wide range

Solubility in H2O highly highly-poorly

Isoelectric point 4.5 2-13

Cleaving enzyme ~100 a few

Amplification >106 no

Modification Methylation Several

Goal of Genomics and Proteomics

• What do biologists want?• Identify proteinssequence of primary structure

and look up genomic and protein database• Characterize proteinsanalyze biologically relev

ant modifications• Look for differetially-expressed proteins as biom

arkers• Look for protein complexes and networksbiolo

gical function(new drug!)

Protein Extract

Analyze spots by AAA SequencingMass spectrometry

Separation based on pI

Separation based on

size

Separate proteins on 2-D gels

Conventional ApproachConventional Approach

1510.010.0 7.57.5 6.36.3 6.06.0 5.85.8 5.55.5 5.25.2 5.05.0 4.04.0 3.03.09.09.0 8.08.0

100100

6060

5050

4040

3535

3030

2525

2020

Mol

ecul

ar W

eigh

tM

olec

ular

Wei

ght

ppII

11

88

77

66

55 1111

44

22 33

3030

99

2020

18181717

1616

15151010

14141313

1919

1212

128128

127127

126126

125125

121121

124124 122122

8787

123123

2828

2727

2626

25252121

2424

2323

2929

22223838

3737

36363434

313135353333

3939

3232

4848 4747

46464545

4141

4444

4343

4949

4242

7272

7878

7777

76767575

7171

7474

7373

6969

4040

7070

6868

6767

666665656161

64646363

7979

6262

6060

58585757

5656

5555

5151

5454

5353

5959

52525050

80808888

129129

8686

8585

8181

8484

8383

8989

8282

9898

97979696

9595

9191

9494

9393

9999

92929090

108108

106106

105105

101101

104104103103

109109102102

100100

118118

117117

116116

107107

111111

114114

113113

119119

112112

110110

115115

151151153153

152152

120120138138

137137

136136 135135

131131

134134

133133

139139

132132

130130

148148 147147

146146

145145

141141

144144

143143

149149

142142

140140

150150

154154

155155

156156

Excise spot;wash; digest

Run 2D gel; Stain/Image

Extract peptides;MALDI-TOF analyzeor qQ-TOF analyze

Database search

Edman DegradationAAA CompositionImmunoblotMS

1045.5584

1183.64981199.6755

1430.7831

1457.6913

1485.72441488.7815

1552.7802

1675.73701876.9218

1894.9346

1909.9414

2167.19222299.1675

2599.2658

1000 1500 2000 2500 Mass (m/z)

Protein ID : Experimental Approach

Peptide-Mass Fingerprinting (PMF)Peptide-Mass Fingerprinting (PMF)

Protein

ProteinSequence

M/Z

M/Z

ProteolyticPeptides

FragmentMass Spectrum

Theoretical FragmentMass Spectrum

TheoreticalProteolytic

PeptidesGLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMKASEDLKKHGATVLTALGGILKKKGHHEAEIKPLAQSHATKHKIPVKYLEFISECIIQVLQSKHPGDFGADAQGAMNKALELFRKDMASNYKELGFQG

GLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMKASEDLK...

Advantages & Disadvantages of 2D gel Advantages & Disadvantages of 2D gel ApproachApproach

• Excellent resolving power• Visual display of protein patterns• Well established technique and Relatively inexpensive to ge

t started with manual techniques• Poor ability to handle certain classes of proteins

• membrane, basic, acidic, high and low molecular weight proteins

• Multiple spots correspond to the same protein or multiple protein products co-migrate to the same spot

• Cannot visualize low abundant proteins; Only abundance proteins are identified

• Time consuming and difficult to automate• Limited recovery of analyte for further analysis• Poor reproducibility; limited dynamic range; certain protei

n stain poorly or not at all.• 2-D gel does not possess the sensitivity and the dynamic ran

ge needed for isolating different proteins

Limitation of 2-D Gel Based Proteomics Platform

• Mouse liver protein database by F. Hoffmann-La Roche Pharmaceutical Research published in Electrophoresis, 2001 showed that analysis of approximately 5800 spots excised from 14 2-D gels resulted in the id of 2,500 proteins which are the products of 328 genes.

• Likewise, in a study published in 2002, only 278 genes whose protein products were identified from rat liver.

Protein Detection MethodsProtein Detection Methods

• Coomassie Blue 0.1 g/band - 1 g/band

• Fluorescent Stain 1 - 10 ng/band

• Silver Stain 1 - 10 ng/band

• 1 ng of a 10kDa 100 femtomoles• 1 ng of a 100kDa 10 femtomoles

High-Performance Liquid Chromatography

LoadingSample

Packing Material

ElutionSample

Impurity 1

Impurity 2, 3

A compound

HPLCLC/MS

Multi Dimensional Liquid Multi Dimensional Liquid ChromatographyChromatography

• Size – Gel filtration columns

• Affinity

• Charge – Ion exchange columns• Hydrophobicity – HIC columns, Reverse phase

Size Exclusion

• Separation of proteins based on their size: The beads are composed of dextran polymers (sephadex), agarose (Sepharose), polyacrylamide (Sephacryl or BioGel P). Each bead contains pores of approximate macromolecule sizes. Larger molecules will travel through less pores, thus migrate faster. Smaller molecules will travel through more pores, thus migrate slower. The molecules passively distribute between the volume outside the porous beads (V

0) and the volume inside the beads (Vi) dependent on their ability to enter the pores. If the total volume of the column is Vt,

Vt = Vo + Vi

Reference: 清華大學 分子與細胞生物研究所暨生命科學系張大慈教授V0

Vi Vt

UV

ab

sorb

ance

Larger Protein

Smaller Protein

Affinity Chromatography

Affinity chromatography makes use of the affinity between a ligand and the protein of interests. Ligands are usually immobilized through covalent bonds on insoluble matrix, such as cellulose or polyacrylamide. The protein of interests become bound to the matrix while other proteins flow through the column. After washing the protein bound to the matrix can be eluted by adding completing groups such as the free ligand, or reagents that disrupt the interactions. Examples of ligand-protein interactions include those between antibodies and antigens, those between Ni+ and poly-histidine tag.

UV

ab

sorb

ance Free ligand concentration

protein

ligand

Ion Exchange Chromatography

• Electrostatic properties of a protein determine the type of ion exchange resins it interacts with. In principle:

– Protein is positively charged if solution pH < pI; It should bind to negatively charged resins, or cation exchanger;

– Protein is negatively charged if solution pH > pI; It should bind to positively charged resins, or anion exchanger;

– Note that in practice, protein surface has local charges that may different from total charge of the protein.

• Examples of cation exchangers include : carboxymethyl (CM) and sulfopropyl (SP)

• Examples of anion exchangers include: diethylaminoethyl (DEAE), quaternary amine (QAE)

proteinsurface-

--++

+-

nonpolar

nonpolar

Ion Exchange Chromatography

• Ion exchange chromatography makes use of electrostatic properties of the protein of interests. Charged polymers are usually immobilized through covalent bonds on insoluble matrix, such as cellulose or polyacrylamide. The protein of opposite charge become bound to the matrix while other proteins flow through the column. After washing, the protein bound to the matrix can be eluted by adding salts.

UV

ab

sorb

ance Increasing salt concentration

protein B

Protein B

protein B

Protein B

Protein A

ProteinANa+ SO3+--CM

Hydrophobic Interaction

• Hydrophobic interaction chromatography makes use of the hydrophobic patches on protein and their interactions with hydrophobic resins.

• For instance, phenyl sepharose is a strong hydrophobic resin that is made by covalently attach phenyl group to agarose supporting matrix. Proteins bind to phenyl group by virtue of hydrophobic interactions. Such interactions are most favored in the presence of high salts. Thus a typical procedure for running hydrophobic chromatography is to first bind proteins under high salt condition and then elute the bound proteins by running a salt gradient from high to low concentrations.

decreasing salt concentration

UV

ab

sorb

ance

proteinsurface-

--++

+-

nonpolar

nonpolar

General approachesGeneral approaches

Protein Extract

Chromatographic Fractionation

2-D GelElectrophoresis

1-D GelElectrophoresis

Biological Pre-fractionation of sample(organs, tissues, cell types, subcellular components)

Enzymatic or chemical Digest

Digest

Chromatography

Label with amino acidtargeted affinity reagentssuch as iTRAQTM

Mass spectrometryElectrospray &/or MALDI

Digest

Multidimensional LCDigest

Protein Protein Identification by Peptide Fragment Fingerprinting

Protein

ProteinSequence

M/Z

M/Z

ProteolyticPeptides

FragmentMass Spectrum

Theoretical FragmentMass Spectrum

TheoreticalProteolytic

PeptidesGLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMKASEDLKKHGATVLTALGGILKKKGHHEAEIKPLAQSHATKHKIPVKYLEFISECIIQVLQSKHPGDFGADAQGAMNKALELFRKDMASNYKELGFQG

GLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMKASEDLK...

PeptideFragments

TheoreticalPeptide

Fragments

LFKLFKGHPELFKGHPETLEGHPETLEKPETLEKETLEK...

SampleIons

LINAC

IRPDetector

Accelerator

Accelerator

Ion Mirror

Curtain Gas

Ground

Pusher Plate

Puller Grid

ESI-Q/TOFESI-Q/TOF

MALDI-TOF/TOFMALDI-TOF/TOF

Source #2

x,y Deflectors #2

Reflector

LinearDetector

ReflectorDetector

Source 2FocusingCID cell

TIS

x,y Deflectors #1

Source 1Focusing

Sample plate & stage

V1

V2

Source #1

Laser

nanoRPLC-ESI-Tandem MSnanoRPLC-ESI-Tandem MS

Total Ion Chromatogram

TOF MS Spectrum

MS/MS Spectrum

N- and C-terminal Peptides

N-term

inal

pep

tides

C-te

rmin

al p

eptid

es

Terminal peptides and ion types

Peptide

Mass (D) 57 + 97 + 147 + 114 = 415

Peptide

Mass (D) 57 + 97 + 147 + 114 – 18 = 397

without

N- and C-terminal Peptides

N-term

inal

pep

tides

C-te

rmin

al p

eptid

es

415

486

301

154

57

71

185

332

429

N- and C-terminal Peptides

N-term

inal

pep

tides

C-te

rmin

al p

eptid

es

415

486

301

154

57

71

185

332

429

N- and C-terminal Peptides

415

486

301

154

57

71

185

332

429

N- and C-terminal Peptides

415

486

301

154

57

71

185

332

429

Problem:

Reconstruct peptide from the set of masses of fragment

Mass Spectra

G V D L K

mass0

57 Da = ‘G’ 99 Da = ‘V’LK D V G

• The peaks in the mass spectrum:– Prefix

– Fragments with neutral losses (-H2O, -NH3)

– Noise and missing peaks.

and Suffix Fragments.

D

H2O

Protein Identification with MS/MS

G V D L K

mass0

Inte

nsity

mass0

MS/MSPeptide Identification:

Sequence from ID

Fragments window shows theoretical ions that match to

spectrum

Label spectrum

easily from fragments window

Mascot Search Mascot Search ParametersParameters

Mascot Search ResultMascot Search Result

Quantitative proteomics

isobaric Tag for Relative and Absolute Quantitation • O16/O18

• ICAT• cICAT• iTRAQ• ICPL• 2D-DIGE• 2MEGA-labeling

1. Reporter Group: Fragmentation of reagent produces a charger reporter ion of mass 114, 115, 116, or 117.

2. Balance Group: Balance mass changes in concert with reporter mass to maintain a total mass of 145.

3. Isobaric Tag: Labeling/mixing of up to four samples with each tag produces peptides with identical m/z. No increase in MS complexity.

4. Protein Reactive Group: The reactive group covalently links the iTRAQTM Reagent to the peptides at the free amines (N-terminus and lysine side chain).

Isobaric Tagging - General Method (4-Plex)P E P T I D E

P E P T I D E

P E P T I D E

+

+

+

+

P E P T I D E

-PRG114 31

-PRG115 30

-PRG116 29

-PRG117 28

1347.0 1349.6 1352.2 1354.8 1357.4 1360.0Mass (m/z)

1352.84

-Reporter-Balance-Peptide INTACT- 4 samples identical m/z

MS

114

115

116

117

111.0 112.8 114.6 116.4 118.2 120.0

Mass (m/z)

114

116

115

117

- Reporter ions DIFFERENT

Mix -N H

P E P T I D E114 31-N H

P E P T I D E115 30-N H

P E P T I D E116 29-N H

P E P T I D E117 28 MS/MS

9.0 292.8 576.6 860.4 1144.2 1428.0

Mass (m/z)

8396.7

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sity y8

P

A b2

y10

q,H

72.1 b4

b1

45.1

b7

LT 112.

1

y3 b10

y5 b9

y2 b6

74.1 13

52.8

y6

142.

1

39.0 y4 b

8

y9 y11

- Peptide fragments EQUAL

P E P T I D EP E P T I D E

Precursor Ion Scan Precursor Ion Scan 用途用途•Small Molecule – 在一堆混合物中尋找代謝物 (Metabolite)標的– 在一堆混合物中尋找合成副產物 (side product)標的– 需知道主結構 (指標性子離子 ) 的分子量– 需要High sensitivity, High speed

•Proteomics– 在一堆混合物中尋找PTM標的– 需知道主結構 (指標性子離子 , 如磷酸根 ) 的分子量– 需要High sensitivity, High speed

Scan Precursors Select ProductCAD

Q1 Q3Q2

Only ions passed through Q1 that produce the PTM-specific fragment mass (e.g. 79 for phosphorylation or 204 for glycosylation) will produce signal at the detector

Precursor ion scan of –79Negative ion mode

Full scan MS (400-1700)Positive ion mode

-casein tryptic digest

Precursor ion scan of –79Negative ion mode

Full scan MS (400-1700)Positive ion mode

-casein tryptic digest

Precursor Ion Scanning (PTM-specific scan)

e.g. Identification of Phosphopeptides in a mixture

phosphopeptides

Matrix Crystals

Embedded Proteins

Chip Surface

Surface Enhanced Laser Desorption/Ionization (SELDI)

• Desorption/ionization method of non-volatile compounds in which the sample presenting surface plays an active role in the extraction, presentation, structural modification, amplification, and/or ionization of a given sample

Invented by T. William Hutchens and Tai-Tung Yip in early 1990s

ProteinChip Technology: Protein Binding

• Crude sample is placed (and processed) on a ProteinChip Array

• Proteins bind to chemical or biological “docking sites” on the ProteinChip surface

ProteinChip Arrays:Available proprietary

surfaces

Hydrophobic Ionic

Chemical Surfaces

Antibody DNA Enzyme Receptor

Biochemical Surfaces

Drug

IMAC

Si

HOOH

Si

OH

SiSi

HO

Hydrophilic

My+ My+

ProteinChip Technology: SELDI TOF-MS Detection

• Retained proteins are “eluted” from the ProteinChip Array by Laser Desorption/Ionization

• Ionized proteins are detected and their mass accurately determined by Time-of-Flight Mass Spectrometry

Inte

nsi

ty

Molecular Mass (Da)

Dete

ctor

Dete

ctor

Laser

TOF-MS

Data presentation:2500 5000 7500 10000 12500

2500 5000 7500 10000 12500

Ret Map 1-4

Ret Map 1-3

Ret Map 1-4(2)

Ret Map 1-3(2)

comp1Difference map

Patient

Control

GelViewTM

Precursor ion fixed

Product ion fixed

Fragmentation(CAD)

MS/MS Multiple Reaction Monitoring (MRM)

127 Da85 Da68 Da

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