Mass Spectrometry and ProteomicsMass Spectrometry and Proteomics

Professor Xudong YaoBioanalytical Chemistry

Spring 2007

• Proteomics and “-omics”

• Roles of mass spectrometry

• Comparative proteomics

• Chemical proteomics

Protein, Proteome and Systems Biology

DNA RNA ProteinTranscription Translation

Rep

licat

ion

Genome Transcriptome Proteome

Proteomics

Analytical Definition of ProteomicsIdentity, Quantity and Function of All the Proteins in a Mixture

Mass Spectrometry

Objectives of Proteomics

Function• Interaction

• Activity

Quantity

Time

Time-Dependence

Identity

Proteome

Analytical Challenges in Post-genome Research

• Sample complexity– “Peak capacity”– Multi-dimensional separation

• Collective analysis– Not traditional, one-by-one analysis– Sensitive, specific and quantitative– and the answers is …

• Data treatment, analysis and achieving– Hardware– Software

• Researchers of multi-disciplinary training

m / z1 5 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 5 5 0 6 0 0 6 5 0 7 0 0 7 5 0 8 0 0 8 5 0 9 0 0 9 5 0 1 0 0 0

%

0

1 0 0

%

0

1 0 0

T im e50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00 450.00 500.00

%

0

100

A Typical Proteomics ExperimentA Typical Proteomics Experiment

Sample PreparationSample Preparation

Mass SpectrometryMass Spectrometry

BioinformaticsBioinformatics

• Protein/Peptide Chemistry• Enzymology• Separations:

Advanced HPLC and Electrophoresis

• New Ionization techniques• Contemporary & New

Mass Spectrometric Methods

• Applications of Mass Spectrometry andDatabase Searching Tools

BiologyBiology• Protein Posttranslational Modification

Protein PhosphorylationProteolysis

Roles of Mass Spectrometry

The mass analysis processThe mass analysis processas compared to the dispersion of light by a prismas compared to the dispersion of light by a prism

Siuzdak, 2003

Mass Spectrometry

Mass Analyzer Ion Detector

Data System

Ion Source(ESI, MALDI)

Sample Introduction

m/z

Intensity

Mass Spectrum

Vacuum

• Separate ions by mass/charge

• Common types– Quadrupole mass filter (Q), Time-of-Flight (TOF), Ion

Trap (IT), Fourier Transform Ion Cyclotron Resonance (FTICR)

• Tandem mass spectrometry– Spatial, such as Q-q-TOF, TOF-TOF, Q-q-Q– Temporal, such as IT, FTICR– Spatial and Temporal, such as IT-FTICR, Q-q-FTICR,

IT-TOF

Mass Analyzer

Soft Ionizations of Soft Ionizations of BiomoleculesBiomolecules

1. Matrix-Assisted Laser Desorption/Ionization (MALDI)

2. Electrospray Ionization (ESI)

Nobel Prize in 2002

MatrixMatrix--Assisted Laser Desorption/Ionization Assisted Laser Desorption/Ionization (MALDI) Generates Singly(MALDI) Generates Singly--Charged IonsCharged Ions

+

+ +

+

+

+

++

+

+ +

+ ++

+

+

+

Sample probe

Laser beam

Light-absorbing matrixAnalyte molecule

+

++

Analyte ion

Matrix ion

__

_

_

MH+

Electrospray Ionization (ESI) Generates MultiplyElectrospray Ionization (ESI) Generates Multiply--Charged IonsCharged Ions

Tandem MS: Basic ConceptsTandem MS: Basic Concepts

MS-1MS-2

Mass-selectedprecursor

Mass Spectrometry for Proteins and Peptides

• Structural elucidation• Quantitative analysis• Sensitive and automatic

mixture analysis

• Sequencing proteins• Identification of post-

translational modifications• High-order structures and

dynamics of proteins and protein dynamics

M+S++A+++N++++D+++++C++++++H+++++++E++++++++M

MSANDCHEM

UNKNOWN PEPTIDE

Mass Spectrometer

Protein/EST Database

Protein Identification

Comparative Proteomics

Gel or Non-gelLabel or Non-label

Comparative Proteomics

Proteome 2

Stable Isotope Coding

Proteome 1

Relative QuantitationConstant Ratio

Abnormal Ratios

Identification of Proteins

SeparationSampling/Cell Culture

Fractionation/Separation

Mass SpectrometryBioinformatics

Relative Changesin proteins including concentration and composition

1. Tween/DOC Extraction (Nuclear Fraction) (Prolactin)2. Tween/DOC Extraction (Nuclear Fraction) (Control)3. Triton X-100 Extraction (Membrane/Organelle Fraction) (Prolactin)4. Triton X-100 Extraction (Membrane/Organelle Fraction) (Control)5. Digitonin Extraction (Cytosolic Fraction) (Prolactin)6. Digitonin Extraction (Cytosolic Fraction) (Control)7. Protein Ladder

1 2 3 4 5 6 7

30K

50K

Reduce sample complexity at the protein Reduce sample complexity at the protein level: DDF (Differential Detergent level: DDF (Differential Detergent

Fractionation)Fractionation)T47D Cells

Pellet

Digitonin Extract

Digitonin

Supernatant

Pellet

Triton X-100 Extract

Supernatant

Triton X-100

Tween/DOC Extract

T47D Cells

Pellet

Digitonin Extract

Digitonin

Supernatant

Pellet

Triton X-100 Extract

Supernatant

Triton X-100

Tween/DOC Extract

GEL: 2-Dimensional Electrophoresis (2-DE)

A Pandey; M Mann, Nat. Biotechnol. 2000

• Difficulties with: extreme pI proteins, low abundance, proteins, hydrophobic proteins• Inefficient in-gel digestion of proteins for MS analysis • Labor extensive

NONNON--GEL: GEL: MudPITMudPIT Analysis of Protein ComplexAnalysis of Protein Complex

• Easier separation• Easier automation• “Problematic” proteins

– Small, large, hydrophobic, low abundance

• Easier sample preparation for MS analysis

• Computational capacity•• Quantitative capabilityQuantitative capability

Link et al. Nat. Biotechnol. 1999

A Glance at Proteomic BioinformaticsA Glance at Proteomic Bioinformatics

ShotgunShotgun2 2 Collision Induced DissociationCollision Induced DissociationMass SpectrometryMass Spectrometry

In-source Shotgun(CID)

In-collision-cell Shotgun(CID)

ShotgunShotgun22(CID)(CID)

CID of [M+3H]3+

CID of [M+2H]2+

****

11

22

3 3 = 1 + 2= 1 + 2InIn--SourceSource

Comparative Proteomics

Proteome 2

Stable Isotope Coding

Proteome 1

Relative QuantitationConstant Ratio

Abnormal Ratios

Identification of Proteins

SeparationSampling/Cell Culture

Fractionation/Separation

Mass SpectrometryBioinformatics

Relative Changesin proteins including concentration and composition

• Large differences in concentration– Direct ESI/MALDI MS

• Small differences in protein concentration– Stable isotope dilution: inherent choice

Label: MS-Based Relative Quantitation

m/z

Intensity

Introduction of Stable Isotopes

• Criteria for isotope internal standards– Ideally behaving the same before mass analysis

• Metabolic labeling during biosynthesis/bioprocess• Post-biosynthesis/bioprocess labeling: chemical

and enzymatic– Functional groups on side chains: -SH, -OPO3H3

– Termini: N-terminal, C-terminal– Active/Binding sites: Chemical Proteomics

(A1A2A3…An)iH2N COOHCoding Module

Advantages of Modular Design

• Isotope Coding • Universal

•Important to small proteins• Specific• Efficient• Minimal Structural Modification

•Chromatographic co-elution•Stable during separation

• Separation• Portable to all separation platforms,including affinity separation

(A1A2A3…An)iH2N COOHSeparation Module Coding Module

Tryptic Incorporation of Two 18O Atoms into Peptide C-Terminus

EHC

R

CHN

OHC

R

C *OH

O

+ H2*O H2N+

*

Adenovirus as Model System

• Minimal real system– DNA + Proteins

• Known genome/proteome– Predictable proteolytic peptides

• Defined architecture– Predictable protein expression

• Comparative quantitation of Ad2/Ad5 proteins• Dynamic range of 600-fold

– Capsid protein modeling membrane/hydrophobic proteins

• Mutations modeling post-translational modifications

Shenk T. In Fundamental Virology, 1996

CombinedIsotope-Coded Peptides

H218O Trypsin H2

16O

Ad2 Virion

3

Ad5 Virion

1

Most of peptidesAd2:Ad5 = 3:1

MALDI-FTICR MS

Protein Identification and Quantitation

Lys-C

Theoretical MW of Ad Tryptic Peptides

MALDI-FTICR Mass Spectrum of Combined Digests

Rel

ativ

e In

tens

ity

218O

218O

218O1603.83, Protein VII

TTVDDAIDAVVEEAR1597.78, Protein V

TSTEVQTDPWFR

1628.96, Protein V

VLRPGTTVVFTPGER

Controversies and Challengesin Proteolytic Labeling

• Reported controversies in tryptic 18O labeling– One 18O incorporation for K-terminated peptides– Low efficient incorporation of two 18O for short peptides– Two 18O in each new peptides

• Capabilities of endoproteases for 18O labeling– Two 18O incorporation by trypsin, Lys-C, and Glu-C only – One 18O incorporation by chymotrypsin…

• Challenges for automated, large-scale application – Amount and cost of H2

18O

Decoupling Proteolytic 18O Labeling from Protein Digestion

Proteins in solution prior to digestionPeptide labeling in small volume of H2

18OSeparate optimization of digestion and labeling

Automatic, high-throughput, large-scale applications

EHC

R

CHN

OHC

R

CHN

O

NH2

H2*OHC

R

C OH

O

H2*O

H2O

HC

R

C OH

O

+ .

½*

½* .

*

* .HC

R

C OH

O*

* +

HO EHO EHO

EHOEHO

H2*O

Amide Bond Cleavage

Carboxyl Oxygen Exchange

Dissection of Proteolytic Incorporation of Two 18O

HC

R

C OH

O½*

½* + EHO

Yao, Afonso, Fenselau. J. Proteom. Res. 2003, 2, 147.

Molecular Basis for Cleavage and Exchange

Protease catalyzes exchange TWO 18O INCORPORATION

P3 P1P2 P3’P2’P1’

S1S2S3 S3’S2’S1’

Native Peptide Substrate

CONHP5 P4

S4S5

S2’

P1

S1 S3’S1’

COOH

Truncated Peptide Substrate

P3 P2

S2S3

P5 P4

S4S5

Exchange

Cleavage

16O-to-18O Exchange Studied by MALDI-FTICR MS

0.5 min

12.0 min730.34 732.34 734.34

YGGFMR(16O2)

YGGFMR(16O18O)

YGGFMR(18O2)

m/zRel

ativ

e In

tens

ity

Time

Determination of Reaction Initial Rates

),,,,,(][][

4242 MMMIIIIIRR

oototal

oo=

Kinetics Comparison in R- and K-Peptides

YGGFMKYGGFMR

kcat/KM

(µM-1min-1)

KM

(µM)

Kcat

(min-1)

4400±7001300±300

0.64±0.142.6±0.9

2800±3003500±500

Simultaneous Mass Spectrometric Determination ofKinetics for Trypsin-Catalyzed 16O-to-18O Exchange

Complete Exchange for Mixture

Exch

ange

Rat

e

R R

Enzymatic 18O Labeling

• Universal two 18O labeling of proteolytic peptides by protease-catalyzed exchange– Both K- and R-terminated peptides– Chymotrypsin and pepsin for two 18O labeling, in

addition to trypsin, Lys-C, Glu-C, …– Both short and long peptides

• 4 Da mass increase at the C-terminus of proteolytic peptides to be differentiated in mass spectrometry

Mass Spectrometry of Peptide-16O2/18O2 Pairs

• 18O-labeling enabled mass spectrometric quantitation

• Effect of peak resolution on quantitation

• Analysis on different mass analyzer configurations

• More than relative quantitation from differential oxygen labeling

(A1A2A3…An)iH2N COOHCoding ModuleSeparation Module

Relative QuantitationUsing Paired Isotope Clusters

0

25

50

75

100

0

25

50

75

100

I4

I2

I0 M0

M2

M4

Theoretical natural isotopic distribution

Observed isotopic distribution of 1:1 mixture of 18O and 16O samples

Mass/Charge

Rel

ativ

e In

tens

ity

⎥⎥⎦

⎤

⎢⎢⎣

⎡−−⎟⎟

⎠

⎞⎜⎜⎝

⎛+⎟⎟

⎠

⎞⎜⎜⎛

−+=o

4

o

22

o

2

o

2

o

2

o

4

MM

MM

MM

II

MM1

IIRatio

1

5

II

Correlation of ESI Quantitation with Peptide UV Quantitation

0 2 4 6 8 100

2

4

6

8

10Peptide FVNQHLCGSHLVE

MS

Rat

io o

f [18

O]/[

16O

]

slope: 0.94±0.03r2: 0.997

UV Ratio of [18O]/[16O]

Reynolds, Yao, Fenselau. J. Proteom. Res. 2003, 1, 27.

Labeling Consistency

BSA Peptide Sequence

Sequence Position

Unlabeled (I0) Elution Time

(minutes)a

Labeled (I4) Elution Time

(minutes)a Ratio 1b I4/I0c

ACFAVE 589-594 46.48 46.48 0.95 0.87 KKFWGKYLYE 155-164 52.17 52.09 0.84 0.85 TYVPKAFDE 519-527 52.93 52.93 0.99 0.96 DKDVCKNYQE 335-344 58.43 58.43 0.99 0.97 DKGACLLPKIE 196-206 57.36 57.36 0.88 0.87 KQIKKQTALVE 544-554 67.70 67.70 0.90 0.88 LLYYANKYNGVFQE 177-190 75.32 75.32 0.99 1.07 YAVSVLLRLAKE 364-375 77.56 77.48 0.91 0.91 AKDAFLGSFLYE 345-356 88.78 88.78 0.86 0.81 DYLSLILNRLCVLHE 474-488 109.11 109.03 0.85 0.96 Average 0.92 0.92 Standard Deviation 0.06 0.08

Reynolds, Yao, Fenselau. J. Proteom. Res. 2003, 1, 27.

MS2 Scan: Identification

Correlation between MS and Western-Blot Quantification of Thioredoxin

in Doxorubicin-Treated HeLa Cells

data01_fraction7inverserepeat #1862 RT: 62.36 AV: 1 SM: 15B NL: 3.42E6T: + NSI d Z ms [ 665.00-675.00]

666 667 668 669 670 671 672 673 674 675m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

668.8

669.2

669.7

670.2

670.7671.2

data01_fraction7inverserepeat #1863 RT: 62.40 AV: 1 NL: 7.07E6T: + c NSI d Full ms2 669.63@35.00 [ 170.00-1350.00]

200 300 400 500 600 700 800 900 1000 1100 1200 1300m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

889.3760.4

583.5

582.8

890.4

689.3

576.3

1018.4574.4 1165.4

1017.4461.2876.3 1019.4690.4

630.3319.2 762.2500.6275.7 744.11001.3

559.1448.0405.2

ESI-IT-MS Analysis of Anion-X Fraction 7:(Labeling: Dox Treated 16O, Control 18O)

Coomassie-Stain

Western-Blot(Anti-Thioredoxin Ab)

ControlDox

Zoom Scan: Relative Quantification

XC= 4.19Dox

Control

[M+H]+=1337.42K.TAFQEALDAAGDK.L

18O2

16O2

Thioredoxin

16O2: 18O2 ≅ 5:1

11 kDa

ControlDox

[M+2H]+2=668.8[M+2H +18O2]+2=670.7

SDS-PAGE Analysis of Whole Cell-Extracts:

Mr

Courtesy of Dan Clark of Stratagene

Effect of Peak Resolution on 18O/16O Ratio (I)

18O2

1603.83, Protein VIITTVDDAIDAVVEEAR

1597.78, Protein VTSTEVQTDPWFR

1628.96, Protein VVLRPGTTVVFTPGER

18O2

18O2

2.6 ±0.33.3±0.53.0±0.53.7±0.42.9±0.3Relative Intensity

0.10.331460% wt

TerminalVIIIIXVIIIIProtein

MALDI-FTICR

Yao, Freas, Ramirez, Demirev, Fenselau. Anal. Chem. 2001, 73, 2836.

Effect of Peak Resolution on 18O/16O Ratio (II)

1922.7 1926.8

m/z

APC3_Human: 0.94

961.83963.84

m/z

2+

APC3_Human: 0.87

894.3 898.3

m/z

APC2_Human: 0.91

m/z

2112.92116.9

A1AH_Human: 0.94

705.00 706.23

m/z

3+

A1AH_Human: 1.3

447.70 449.68

m/z

2+

APC2_Human: 1.2

MALDI-TOF

ESI-IT

Heller, Mattou, Menzel, Yao. JASMS 2003, 14, 704.

Effect of Isolation Window Width on Quantitation Using 16O/18O y-Ion Pairs on IT MS

984.

60

988.

49

983 985 987 989 991 m/z

984.

62 988.

60

983 985 987 989 991 m/z

689.

85

686 688 690 692 694 m/z

689.

87 691.

82

686 688 690 692 694 m/z

MS

MS/MS

MS

MS/MS

Isolation of Io = 689.93 Isolation of I4 = 691.93

MH22+ MH2

2+

y10+ y10

+

691.

83

Heller, Mattou, Menzel, Yao. JASMS 2003, 14, 704.

Protein Sequence IonsGenerated by Tandem Mass Spectrometry

H2N C C

A1

HN

O

C C

A2

HN

O

C C

A3

HN

O

C C

A4

HN

O

C C

A5

OH

OH H H H H

x1 y1 z1

a4 b4 c4

x2 y2 z2

a3 b3 c3

x3 y3 z3

a2 b2 c2

x4 y4 z4

a1 b1 c1

Mass Analyzer1

Mass Analyzer2

Fragmentingthe Selection Ion

Masses of Amino Acid ResiduesMasses of Amino Acid Residues

Yao, Freas, Ramirez, Demirev, Fenselau. Anal. Chem. 2001, 73, 2836.

Quantitation Based on MS/MS Spectrum (y-Ions)

m/z

MTVEPGLEPEVR

678 680 682 6840

100

Rel

ativ

e In

tens

ity

m/z

16O2

18O2

350 400 450 500 550 600 650 700 750

0

20

40

60

80

100

Rel

ativ

e In

tens

ity

H2N C C

A1

HN

O

C C

A2

HN

O

C C

A3

HN

O

C C

A4

HN

O

C C

A5

OH

OH H H H H

y1

b4

y2

b3

y3

b2

y4

b1

16O/18O Paired Peptides Facilitate and Validate Peptide Sequencing

N-terminal mass470.1 Da

QqTOF MS/MS

IT MS/MS

m/z400 500 600 700 800 900 1000 1100 1200

Q D A Y V SC-terminal mass575.3 Da

Rel

ativ

e In

tens

ity

Advantages of Modular Design

• Isotope Coding • Universal

•Important to small proteins• Specific• Efficient• Minimal Structural Modification

•Chromatographic co-elution•Stable during separation

• Separation• Portable to all separation platforms:LC/LC, solution IEF, affinity separation

(A1A2A3…An)iH2N COOHSeparation Module Coding Module

Protein Pools of Digitonin Extract of MCF-7 Cells

Modified according to “Ramsby, Makowski Method Mol. Biol., 112, 1999.

• Digitonin fraction– cytosolic – soluble cytoskeletal proteins

• Properties– functional proteins– soluble proteins

Cancer Cells

Pellet

5,000xg

Digitonin Extract

Digitonin Extraction

Supernatant

120,000xg

C4 Fractionation

LC-MS of Peptides from MCF-7 Digitonin Fraction

4 0 6 0 8 0 1 0 0 1 2 0

Rel

. Int

.

Base peak chromatography of total peptide mixture

Time (min)

Rel

. Int

.

0.5 1.0 1.5 2.0 2.5 3.00

10

20

30

40

Freq

uenc

y

MelR/WT

• Protein Ratio (MelR/WT) = 1.1• I0/I4 Ratio (MelR/WT) = 1.1 ± 0.3• 83% (184/223) peptides in 1.1 ± 0.3

m/z600 650 700 900 950 1000

TOF MS at 78 min

m/z600 650 700 900 950 1000

TOF MS at 78 min

Yao, Fenselau. ASMS Annual Conference, 2001

Protein Expression Changes in MCF-7 CellsUpon Acquisition of Melphalan Resistance

[Most Proteins in a Ratio (MelR/WT) of 1.1]

784 786 788

(K)LLPQLTYLDGYDR(E)PHAPI2b /April proteinpI = 4.0MelR/WT = 2.0

654 656 658 660

(R)GIVTNWDDMEK(I)602308605F1 NIH_MGC_88 Homo sapiens cDNA cloneMelR/WT = 2.6

m/z

Re l

ati v

e In

ten s

ity

Yao, Fenselau. ASMS Annual Conference, 2001

Analysis of Human Plasma Sample: Example 1

Abundance Protein Depletion

Denaturing SEC Small Proteins

Large Proteins RPFraction X

Tryptic Digestion

Human Plasma

1x Peptide Pool 1 1x Peptide Pool 2

Enzymatic 16O-Labeling Enzymatic 18O-Labeling

2x MG 16O-Peptides 1x MG 18O-Peptides

Combine Differentially-Labeled Peptides

µ-RP-HPLC ESI-MS/MSMALDI-MS/MS

SCX

Heller, Mattou, Menzel, Yao. JASMS 2003, 14, 704.

LC-MALDI & LC-ESI MS Analysis ofDifferentially 18O/16O-Labeled Peptides

Present in Human Plasma

LC-nanoESI-MS (QTOF)748.43

752.47

570.29 571.63

748.42

752.42

570.32 571.65

LC-ESI-MS (IT)

LC-MALDI-MS (TOF)1708.5 1712.5

748.3

752.3

Rel. Int.

m/zHeller, Mattou, Menzel, Yao. JASMS 2003, 14, 704.

Analysis of Human Plasma Sample: Example 2

Qian et. al., Smith MCP 2005, March 7

Coupling 16O/18O-Labeling and Solution Isoelectric Focusing for Peptide Analysis

An, Fu, Gutierrez, Fenselau. J. Proteom. Res. 2005, 4, 2126.

─ Comparative investigation of insoluble nuclear subproteome─ Sample separation after combining differentially-labeled peptides

Isotope-Coded Affinity Tag (ICAT)

• Unique Chemistry for -SH• Affinity Tag• Isotope-Coded Linker

Gygi et al. Nat Biotechnol. 1999

Assembling Separation Module and Coding Module

O -16

O -18

Mass/Charge

Rel

ativ

e In

tens

ity

O

O

NH O O NH

OS

NHNH

I

H218/16O

16/18O2

16/18O216/18O2

16/18O2

16/18O2

CH2

S

OH

O16/18

O

Biotin

(A1A2A3…An)iH2N COOHCoding ModuleSeparation Module

ProteinGlu-C

16/18

–16O2–18O2

FVNQHLCGSHLVE

Biotin

16O/18O-Labeling and Affinity Enrichment to Quantitate Proteins

Liu, Qian, Strittmatter, Camp, Anderson, Thrall, Smith. Anal. Chem. 2004, 76, 5345.