Welcome to the world of mass spectrometrymass spectrometry ...

Welcome to the world of mass spectrometrymass spectrometry-based proteomics!p

Xi l Zh Ph DXiaolu Zhao, Ph.D.Room 6016, CLSWuhan UniversityWuhan University

从"基因"到"蛋白"

基因数

线虫 19,000

果蝇 13,500

拟南芥 27 000拟南芥 27,000

人类 ~20,500人类 20,500

从"基因"到"蛋白"

基因数

线虫

基于质谱技术的蛋白质组学 - 全局性研究线虫 19,000

果蝇 13,500 蛋白质的表达水平

拟南芥 27,000

人类 20 500

蛋白质的表达水平

蛋白质翻译后修饰

蛋白质相互作用人类 ~20,500

PROTEIN (蛋白质)DNA RNA蛋白质相互作用

PROTEIN (蛋白质)DNA RNA

Protein and proteomicsProtein and proteomicsMass spectrometry basicsM t t f t iMass spectrometry for proteomicsQuantitative proteomicsQ pHuman Proteome and more

P t i d t iProtein and proteomics

Amino acid structure

R

Side chain (R)

HN C

R

C OHH

N C

H

C OH

O

Amino group

Carboxyl groupgroup group

Protein Backbone

H HR’ R’’H R H

C N

H

C C N

R

C C

R

OH OH OH

H

C

R

N

O H O H OH H

H O H O

HH

H HR’ R’’

H2O H2O

H R

C

O

N

H

C C

O

N

H

C C

O

OH

HHCN

O H O H O

Amide bond

HH

Amide bond

Amino Acid Residue

H

C N

H

C C N

R’

C C

R’’

OH

H

C

R

N C

O

N

H O H OHHCN

H R’HN

HC C

OH O

Amino acid residue structure

What is a proteome?What is a proteome?

The proteome is the entire set of proteins expressed by a genome, cell, tissue or organism at a certain time Moreorganism at a certain time. More specifically, it is the set of expressed proteins in a given type of cell or organism, p g yp g ,at a given time, under defined conditions.

What is Proteomics?What is Proteomics?

Systematic (large scale) study ofSystematic (large scale) study of protein properties to obtain a global integrated view of diseaseglobal, integrated view of disease processes, cellular processes and networks at the protein level

What is Proteomics?What is Proteomics?

“Proteomics includes not only the identification and quantification of proteins, but also the determination of theirbut also the determination of their localization, modifications, interactions, activities, and, ultimately, their function.”

-Stan Fields in Science 2001-Stan Fields in Science, 2001.

Every set of proteins of same modification can be called a proteomep

Phosphorylation Phosphorylome

Acetylation

Ubiquitination

Acetylome

Ubiquitinome

Methylation

GlcNAclation

Methylome

GlcNAclomeGlcNAclation GlcNAclome………

The tasks in proteomicsThe tasks in proteomics

Detection What and WhereWhat and Where

Quantification Compare dynamicsCompare dynamics

Technologies for ProteomicsTechnologies for Proteomics

Adapted from Mallick and Kuster, Nature Biotechnology, 28:695-709, 2010

Conceptual Organization of Proteomic ExperimentsConceptual Organization of Proteomic ExperimentsConceptual Organization of Proteomic ExperimentsConceptual Organization of Proteomic Experiments

Adapted from Mallick and Kuster, Nature Biotechnology, 28:695-709, 2010

Challenges in proteomicsg p

DNA: 4 basesP t i 20 i id + PTMProtein: 20 amino acids + PTM

DNA: similar biochemical property

Protein: i 450average size 450 aa

wide range of solubilityPTMdegradationdegradation

The Human Plasma ProteomeThe Human Plasma Proteome

overwhelming dynamic range of at least 10 orders of magnitude

Proteomics - ChallengesProteomics Challenges

• Sample• Sampling Handling• Complexity Reduction• Chromatography• Mass Spectrometry• Bioinformatics

( )Aebersold (2009) Nature Methods, 6, 411

Why proteomics is a complement or y p palternative to mRNA-based measurements?• In areas in which microarray measurement is not

feasible. Example: blood.

measurements?

• The proteomic measurement already delivers the desired end point, namely the protein expression level of a gene of interestof a gene of interest

• It is not limited to expression profiling of the whole cell. Cellular compartments and organelles and their time-p gresolved dynamics are readily accessible to this technology

f f• The large-scale measurement of protein modifications and their quantitative changes upon perturbations to the cellthe cell.

Figure： Quantitative Proteomics versus TranscriptomicsFigure： Quantitative Proteomics versus Transcriptomics

(A) Overlap between the proteins identified in the example given in Figure 1 (blue) and the messages with “present call” (that is, significantly different from zero signal) in a quadruple microarraysignificantly different from zero signal) in a quadruple microarray experiment of the normal HeLa cell proteome (green).

(B) “Label-free” quantitation of about 4000 proteins identified in brain tissue samples from two separate individuals. Mass spectrometric intensity counts (added peptide signals) from the two separate runs are plotted against each other.

Mass spectrometry basicsMass spectrometry basics

Mass spectrometry to proteomics isMass spectrometry to proteomics isMass spectrometry to proteomics is Mass spectrometry to proteomics is like PCR to genomicslike PCR to genomicsgg

MS PrinciplesMS Principles

• Different elements can be uniquely identified by their mass

MS PrinciplesMS Principles

• Different compounds can be uniquely identified by their mass

Butorphanol L-dopa EthanolNOH

-CH2-

CH CH NHCOOH

HO CH3CH2OH

HO

-CH2CH-NH2HO

HOHO

MW = 327.1 MW = 197.2 MW = 46.1

What is Mass Spectrometry?What is Mass Spectrometry?

Mass spectrometry is a powerful technique forMass spectrometry is a powerful technique for chemical analysis that is used to identify unknown compounds to quantify known compounds and tocompounds, to quantify known compounds, and to elucidate molecular structure.

Wh t d t t d ?What does a mass spectrometer do?

1. It measures mass better than any other technique.

2. It can give information about chemical structuresstructures.

3. It generate spectrum by separating gas phase ions of different mass to charge ratio (m/z)

m=molecular or atomic massz = electrostatic charge unit

Typical Mass Spectrum

Ch t i d b h• Characterized by sharp, narrow peaksX i iti th / tisi

ty

3.3E+

80

90

100

Voyager Spec #1[BP = 2185.1, 32728]

2185.1265

1746 9323 • X-axis position: the m/z ratio of a given ion H i ht f k l tie

inte

ns

50

60

70

80

% In

tens

ity

1746.9323

3442.7118

• Height of peak: relative abundance of a given ion

Rel

ativ

e

10

20

30

40 1303.6221

1626.7960

2975.45221052.6278

1903.0161969.5085 1711.8485 2207.0981

1068.61622647.17881726.8255

2189.1376 2989.47071640.8147666.0449 3446.73661894.93651347.7358877 0664 2140 1285 2979 4543

m/z • Peak intensity indicates the ion’s ability to “fly” (some fly

R

499.0 1399.2 2299.4 3199.6 4099.8 5000.0Mass (m/z)

00

1347.7358877.0664 2140.1285 2979.45432663.1801 3398.6503606.2721 1886.8093 2341.2018

ion’s ability to “fly” (some fly better than others)

Multiple Chargingp g g

Calculating mass-to-charge ratio (m/z)Consider a peptide with MW of 10000

With ESI-MS, charges by H+ additionM + nH+ MnHn+

Resultant ions formed are:When z = 1 m/z = (10000+1)/1 = 10001When z = 1 m/z = (10000+1)/1 = 10001When z = 2 m/z = (10000+2)/2 = 5001Wh 3 / (10000+3)/3 3334 3When z = 3 m/z = (10000+3)/3 = 3334.3When z = 4 m/z = (10000+4)/4 = 2501When z = 5 m/z = (10000+5)/5 = 2001

Figure from The Expanding Role of MS in Bio-technology – G . Siuzdak

Stable isotopes of most abundantStable isotopes of most abundant elements of peptides

Element Mass AbundanceH 1.0078 99.985%H 1.0078

2.014199.985%0.015

C 12.000013 0034

98.891 1113.0034 1.11

N 14.003115 0001

99.640 3615.0001 0.36

O 15.994916.9991

99.760.04

17.9992 0.20

Mass spectrum of peptide with 94 C-atoms (19 amino acid residues)(19 amino acid residues)

“

1981.84No 13C atoms (all 12C)

“Monoisotopic mass”

1982.84 One 13C atom

1983.84Two 13C atoms

Monoisotopic massMonoisotopic massMonoisotopic masspcorresponds tolowest mass peak

When the isotopes are clearly resolved the monoisotopic mass is used as it is the most accurate pmeasurement.

Average massAverage mass

AAverage mass corresponds to the centroid of thecentroid of the unresolved peak cluster

When the isotopes are not resolved, the centroid of the envelope corresponds to the weighted average of all the the i t k i th l t hi h i th thisotope peaks in the cluster, which is the same as the average or chemical mass.

Mass Calculation (Glycine)Mass Calculation (Glycine)

NH CH COOHNH2—CH2—COOH Amino acid

R1—NH—CH2—CO—R3 Residue

Glycine Amino Acid MassMonoisotopic Mass1H = 1.00782512C 12 00000

Glycine Amino Acid Mass5xH + 2xC + 2xO + 1xN= 75.032015 amu12C = 12.00000

14N = 14.0030716O = 15 99491

Glycine Residue Mass3xH + 2xC + 1xO + 1xN

57 021455O = 15.99491 =57.021455 amu

Amino Acid Residue MassesAmino Acid Residue Masses

Monoisotopic Mass

Glycine 57.02147Al i 71 03712

Aspartic acid 115.02695Gl t i 128 05858

Monoisotopic Mass

Alanine 71.03712Serine 87.03203Proline 97 05277

Glutamine 128.05858Lysine 128.09497Glutamic acid 129 0426Proline 97.05277

Valine 99.06842Threonine 101.04768

Glutamic acid 129.0426Methionine 131.04049Histidine 137.05891

Cysteine 103.00919Isoleucine 113.08407L i 113 08407

Phenylalanine 147.06842Arginine 156.10112T i 163 06333Leucine 113.08407

Asparagine 114.04293Tyrosine 163.06333Tryptophan 186.07932

Amino Acid Residue Masses

Average Mass

Glycine 57.0520Al i 71 0788

Aspartic acid 115.0886Gl t i 128 1308

Average Mass

Alanine 71.0788Serine 87.0782Proline 97 1167

Glutamine 128.1308Lysine 128.1742Glutamic acid 129 1155Proline 97.1167

Valine 99.1326Threonine 101.1051

Glutamic acid 129.1155Methionine 131.1986Histidine 137.1412

Cysteine 103.1448Isoleucine 113.1595L i 113 1595

Phenylalanine 147.1766Arginine 156.1876T i 163 1760Leucine 113.1595

Asparagine 114.1039Tyrosine 163.1760Tryptophan 186.2133

Important performance factorsp p

Mass accuracy: How accurate is the mass measurement?mass measurement?

Resolution: How well separated are theResolution: How well separated are the peaks from each other?

Sensitivity: How small an amount can be analyzed?be analyzed?

ResolutionResolutionm

mR

分辨率：分开两个邻近质量峰的能力，高分辨率将目

标物与复杂基质分离，排除干扰，确保质量精度。

m1 m2

未分开分辨率差

部分分开分辨率中等

全分开分辨率高

What if the resolution is not so good?What if the resolution is not so good?

PBetter Poorer resolutionresolution

6130 6140 6150 6160 6170Mass

At lower resolution, the mass measured is the average mass.

Glossary:mass accuracy

• Mass accuracy: The absolute Mass accuracy: The absolute deviation between measured mass f f ll from true mass of an ion, typically expressed as an error value in ppm expressed as an error value in ppm (parts per million).

1000±0.1 = 0.01%= 100 ppm= 100 ppm

Mass measurement accuracy depends on resolutiony p

R l ti 18100

High resolution means better mass accuracy

6000

8000Resolution =18100

15 ppm error

4000

6000

Cou

nts Resolution = 14200

24 ppm error

2000Resolution = 4500

55 ppm error

0

2840 2845 2850 2855

Mass (m/z)( )

Wh t d ll MS i t t h i ?Wh t d ll MS i t t h i ?What do all MS instrument have in common?What do all MS instrument have in common?

Sample introduction Separate Count ions

Collect resultsintroductionIonization

Minimize collisions, interferences

masses Collect results

Sample IntroductionSample Introduction

High Vacuum System

I M DInlet IonSource

Mass Analyzer Detector

DataSystem

HPLCGCFlow injectionSample plate

Ion SourceIon Source

High Vacuum System

I M DInlet IonSource

Mass Analyzer Detector

DataSystem

MALDIESIFABLSIMSEICI

Mass Spec Principlesp p

SampleSample

++_

Ionizer Mass Analyzer Detector

• Find a way to “charge” an atom or molecule

• Place charged molecule in a magnetic field or subject it to an

• Detect ions using microchannelj

electric field and measure its speed or radius of curvature

microchannel plate or photomultiplier tuberelative to its mass-

to-charge ratiotube

Nobel Prize in Chemistry 2002y

"f th d l t f th d f "for the development of methods for identification and structure analyses

of biological macromolecules" of biological macromolecules

"for their development of soft desorption ionisation methods for mass spectrometric NMRionisation methods for mass spectrometric

analyses of biological macromolecules" NMR

John B. Fenn Koichi Tanaka

ESI MALDIESI MALDI

b. 1959b. 1917

Matrix-assisted laser desorption/ionization (MALDI) 基质辅助激光解析电离

Pulsed laser

(MALDI) 基质辅助激光解析电离

Pulsed laser

Sample plate IonsSample plate

Extraction

Ions

Extraction grid

• Absorption of UV radiation by chromophoric matrix and p y pionization of matrix

• Dissociation of matrix, phase change to super-compressed fgas, charge transfer to analyte molecule

• Expansion of matrix at supersonic velocity, analyte trapped in expanding matrix plume (explosion/”popping”)

Adopted from Nature, 422,200 (2003)

in expanding matrix plume (explosion/ popping )

MALDIMALDIMALDIMALDI

MALDIMALDIMALDIMALDI

384 spotsp

Matrix for MALDI ToFMatrix for MALDI ToFMatrix for MALDI ToFMatrix for MALDI ToF

• 2,5-dihydroxybenzoic acid (DHB)• α-cyano-4-hydroxy-cinnamic acidy y y• 3,5-dimethoxy-4-hydroxycinnamic acid

(sinapinic acid)(sinapinic acid)• Specific compounds for glycoprotein etc

Example of a MALDI-TOF Mass Spectrump p

Voyager Spec #1[BP = 2185.1, 32728]

3.3E+4

90

100 2185.1265

70

80 1746.9323

50

60

% In

tens

ity

3442.7118

30

40 1303.6221

1626.7960

2975.45221052.6278

10

20 1903.0161969.5085 1711.8485 2207.09811068.6162

2647.17881726.82552189.1376 2989.47071640.8147666.0449 3446.73661894.93651347.7358877.0664 2140.1285 2979.4543

2663.1801 3398.6503606.2721 1886.8093 2341.2018

499.0 1399.2 2299.4 3199.6 4099.8 5000.0Mass (m/z)

00

Electrospray Ionization (ESI)Electrospray Ionization (ESI)p y ( )p y ( )Atmosphere Vacuum

Li id SIons

p

Liquid chromatography

Spray needle

Nozzle SamplingNozzle Samplingcone

质谱技术的核心——质量分析器质谱技术的核心 质量分析器真空系统真空系统

大气压检测器检测器

数据处理数据处理系统系统质量分析器离子化方式

进样系统(LC或者直

大气压检测器检测器

系统系统质量分析器离子化方式( 或者接进样)

杆四级杆 (Q)离子阱/线性离子阱 (IT/LTQ)飞行时间 (TOF)

低分辨质谱

中高分辨质谱飞行时间 (TOF)傅立叶变换离子回旋共振 (FTICR)静电场轨道阱 (Orbitrap) 超高分辨质谱

中高分辨质谱

( p)

Analyzer TypesAnalyzer Types

• What is the analyzer?Analyser is the section of instrument that separates ions of different m/zthat separates ions of different m/z• Many Different technologies:Many Different technologies:Magnetic Sector, Quadrupole, Ion Trap, ToF

The quadrupole consists of four parallel metal rods

Only ions of a certain m/q will reach the detector for a given ratio of voltages:other ions have unstable trajectories and will collide with the rods.

This allows selection of a particular ionThis allows selection of a particular ion, or scanning by varying the voltages.

Obtains a mass spectrum by sweeping across the entire mass range

MALDI-TOF SMass Spectrometry

B k A t flBruker Autoflex

Now available as Tof/ToF

Easy to use – walk up use after training.g

Highly automated

N b d fNow can be used for imaging of Tissue samplessamples

QQ TOFTOFQQ--TOFTOF

NANOSPRAY TIP

MCPDETECTORDETECTOR

TOFHEXAPOLECOLLISIONCELL

HEXAPOLE

PUSHER

TOF

IONSOURCE

CELL

HEXAPOLE

QUADRUPOLEREFLECTRONSKIMMER

SOURCE

超高分辨质谱——傅立叶变换FTFT-ICR

傅立叶变换离子回旋共振Orbitrap

静电场轨道阱

zmk/

zm

B/

傅立叶变换质谱——当离子进入质量分析器后，以和质荷比相关的特征频率作轨道方式运动，

随后离子被激发到其固有的轨道半径旋转，由收集电极检测随时间变化的镜像电流。时域信

号经FT变换器得到频率域信号 继而转化为质谱图号经FT变换器得到频率域信号，继而转化为质谱图。

独一无二的Orbitrap技术中心电极

外纺锤电极外纺 极

30年来唯一基于全新理论的质量分

析器，真空度比传统质量分析器高3

个数量级以上，稳定性极强。

Orbitrap——逐渐成为质谱分析的金标准

Orbitrap性能9年来飞速增长

500000H

M) 400000

450000Orbitrap

Tof / QTofOrbitrap Fusion

ion

(FW

H

300000

350000

s re

solu

t

200000

250000 Orbitrap Elite QE HF

Mas

s

50000

100000

150000

LTQ Orbitrap

QE

Bendix Tof0

50000

1955 1965 1975 1985 1995 2005 2015

First Q-Tof

Time progression (year)

性能卓越的Q-Exactive系列质谱仪性能卓越的Q Exactive系列质谱仪

Q-Exactive (2011)尖端四极杆

Q-Exactive Plus (2013)尖端四极杆

超高场Orbitrap

Q-Exactive HF (2014)高 p

高灵敏度

四极杆 Orbitrap高灵敏度

高选择性

高动态范围

高分辨率

高质量精度

How do mass spectrometers get their names?

Types of ion sources:

g

• Electrospray (ESI)

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

Types of mass analyzers:

Q d l (Q d Q)• Quadrupole (Quad, Q)

• Ion Trap

• Time-of-Flight (TOF)

Either source type can work with either analyzer type: “MALDI-TOF,” “ESI-Quad.”. Analyzers can be combined toMALDI TOF, ESI Quad. . Analyzers can be combined to create “hybrid” instruments. ESI-QQQ, MALDI QQ TOF, Q Trap

Summary: acquiring a mass spectrum

Ionization Mass Sorting (filtering) Detection

y q g p

Ionization Mass Sorting (filtering)

Ion

Detection

Ion S Mass Analyzer DetectorSource

Detect ionsForm ions

(charged molecules)Sort Ions by Mass (m/z)

(charged molecules)

Inlet • Solid• Liquid

100

75

50

25q• Vapor 1330 1340 1350

0

Mass Spectrum

Mass spectrometry for proteomics

Breaking Protein into PeptidesBreaking Protein into Peptides

Name Cleave Don't cleave N or C termName Cleave Don t cleave N or C termTrypsin KR P CTERMArg-C R P CTERMAsp-N BD NTERMAsp-N BD NTERMAsp-N_ambic DE NTERMChymotrypsin FYWL P CTERMCNBr M CTERMCNBr M CTERMFormic_acid D CTERMLys-C K P CTERMLys-C/P K CTERMLys C/P K CTERMPepsinA FL CTERMTryp-CNBr KRM P CTERMTrypChymo FYWLKR P CTERMTrypChymo FYWLKR P CTERMTrypsin/P KR CTERMV8-DE BDEZ P CTERMV8-E EZ P CTERMV8 E EZ P CTERMCNBr+Trypsin M CTERM

KR P CTERM

Protein ID: Two Main ApproachesProtein ID: Two Main ApproachesProtein ID: Two Main ApproachesProtein ID: Two Main Approaches

• Peptide Mass Fingerprinting (PMF)

• Tandem Mass Spectrometry (MS/MS)p y ( )

Peptide Mass Fingerprinting (PMF)p g p g ( )

Generalized Protein Identification by Generalized Protein Identification by PMFPMF

Spot removed f l

Trypsin digest MS1 of tryptic peptidesfrom gel peptides

y

MATCH

Libr

ary

Artificial spectra built

Artificially trypsinated

Database of sequencesspectra built trypsinated (i.e. SwissProt)

Principles of Fingerprintingp g p gSequence Mass (M+H) Tryptic Fragments

>Protein 1acedfhsakdfqeasdfpkivtmeeewe

q ( ) yp g

4842.05

acedfhsakdfgeasdfpki t d d f ksdfpkivtmeeewe

ndadnfekqwfe

>P t i 2

ivtmeeewendadnfekgwfe

>Protein 2acekdfhsadfqeasdfpkivtmeeewe

4842.05

acekdfhsadfgeasdfpkivtmeeewenk

nkdadnfeqwfe

>Protein 3

dadnfeqwfe

acedfhsadfgekacedfhsadfqekasdfpkivtmeeewendakdnfeqwfe

4842.05

acedfhsadfgekasdfpkivtmeeewendakdnfegwfeq dnfegwfe

Principles of Fingerprintingp g p gSequence Mass (M+H) Mass Spectrum

>Protein 1acedfhsakdfqeasdfpkivtmeeewe

q ( ) p

4842.05 sdfpkivtmeeewendadnfekqwfe

>P t i 2>Protein 2acekdfhsadfqeasdfpkivtmeeewe

4842.05

nkdadnfeqwfe

>Protein 3acedfhsadfqekasdfpkivtmeeewendakdnfeqwfe

4842.05

q

Can We Do Better?

Single Stage MS Tandem MS

Protein Identification StrategyProtein Identification Strategy

II

f gyf gy

*IIPeptidesPeptides

12 14 16

Time (min)ProteinProtein1D, 2D, 3D peptide separation1D, 2D, 3D peptide separation

*

Protein Protein mixturemixture

Q2Q2 Q3Q3

IIII200 400 600 800 1000 1200

m/zm/zTandem mass spectrumTandem mass spectrumQ1Q1 Q2Q2

Collision CellCollision CellQ3Q3

Correlative Correlative

Tandem mass spectrumTandem mass spectrumQ1Q1

IIIIII sequence database sequence database searchingsearching

200 400 600 80010001200m/zm/z

200 400 600 80010001200m/zm/z

TheoreticalTheoretical AcquiredAcquiredProtein identificationProtein identification

m/zm/zm/zm/z

Cleavages Observed in MS/MS of Peptides

yn-i zn-ilow energy

xn-i

n i

HN CH CO NH CH CO NH-HN--CH--CO--NH--CH--CO--NH-

Ri CH-R’Ri CH-R

R”ai

ci

Rbi

Fragmenting a Peptideg g p

http://www.proteomecenter.org/course/2005.jan.eng.pdf

MS/MS Peptide Fragmentationp g

Ala-Gly-His-Leu-….Phe-Glu-Cys-Tyr

b1 y1 b2 y2 b3 y3 b4 y4 b5 y5

Example of a ms/ms spectrump p

100YADSGEGDFLAEGGGVR

80

ce

XF D G

YADSGEGDFLAEGGGVR

40

60

bund

anc

EA E

GS

20

40

lativ

e A

b S D

400 600 800 1000 1200 1400 1600

0Re

m/z

Mascot MS/MS Form

Mascot MS/MS Output

Mascot MS/MS Output

MS/MS vs PMFS/ S sAdvantages Disadvantages

• Provides precise sequence-specific data

• Requires more handling, refinement and sample

Advantages Disadvantages

specific data• More informative than PMF

methods (>90%)

refinement and sample manipulation

• Requires more expensive and ( )• Can be used for de-novo

sequencing (not entirely d d t d t b )

complicated equipment• Requires high level expertise

dependent on databases)• Can be used to ID post-

trans modificationstrans. modifications

Quantitative proteomics

Quantitative proteomicsQuantitative proteomics

• The goal of a quantitative proteomics analysis is to determine the changes in protein expression in a given cell from a given organism when subjected to a stimulus. g g j

Label-free Quantitation

Two examples of label-free quantitation：

XIC – extracted ion chromatogramSC – spectral counting

Avoid isotopes but instrumentation needs to be d iblvery reproducible

Label-free Quantitation - XIC

if 660.96 is identified by MS/MS as a peptide coming from protein X

XIC of 660.96

Only one peak because none of the other peptides have a molecular mass of 660.96Find the peak area

Repeat the same process for all the other peaks B, C and DSum all the peak areas in the XICsConcentration of protein X proportional to total area (need an internal standard)

Label-free Quantitation – SC

MS of each peaks

MS of peak B

Peak at 2116 8 is 3rd highest soCount all the MS/MS spectra

hi h f idPeak at 2116.8 is 3 highest so selected for MS/MS

which came from a peptide which can be identified as coming from protein X (S t l C t)(Spectral Count)

Spectral count proportional

90

Use e.g. protein Prospector to d t i th t thi tid i

to protein concentration (need internal standard)

determine that this peptide sequence is derived from protein X

G

Stable Isotope Labeling Strategies

Metabolic stablei t l b liIN

LA

BEL

ING

isotope labeling

PRO

TEI

Digest

LEC

TIO

ND

ATA

CO

LD

YSIS

Mass spectrometryMass spectrometry

ATA

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SILAC – stable isotope labelling of amino id i ll ltacids in cell culture

Grow cells in media containing isotopically labelled amino acidsGrow cells in media containing isotopically labelled amino acids

O

Typically e.g.

L 4 lkl D 4 b it ti +4 itNH2

NH2OH

Lys4 – alkly Dx4 subsitution – +4 unitsArg6 – 13Cx6 subsitution – +6 unitsLys8 - 13Cx6 + 15Nx2 substitution +8 unitsArg10 13Cx6 + 15Nx4 substitution +10 units NH

O

NH

NH2

OHArg10 - 13Cx6 + 15Nx4 substitution +10 units NH NH

NH2

OH

Labelling arginine and lysine to ensure all tryptic peptides are labelled (Trypsin cuts at K or R)labelled (Trypsin cuts at K or R)

SILAC – stable isotope labelling of i id i ll ltamino acids in cell culture

L t t t i tLyse, extract protein, separatetrypsin digest, MS

SILAC mouse

SILAC ?SILAC SILAC mouse?

Protein Chemical labeling

G

Stable Isotope Labeling Strategies

Isotope taggingIN L

AB

ELIN

G

Isotope taggingby chemical

reactionLabel

PRO

TEI

Digest Digest

Digest

LEC

TIO

N Label Label

DAT

A C

OL

DYS

IS

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iTRAQ Isobaric Tag for Relative and Absolute Quantification

Reacts with NH groupsReacts with NH2 groups

NNH R

Adds tag of mass 145 to N

NCH3

O OAdds tag of mass 145 toterminal NH2 groups and lysines

NCH2

+MS/MS Fragmentation

97

NCH3Rest of molecule +

Reporter ion

iTRAQ

NO

N

OMw = 2813C x 3 15N x 1

N

NCH3

O

N

O O

OC18O13C x 2

3 O

Produces an ion of Mw = 117f f

N

NCH3

O

N

Oafter fragmentation

O13CO

CH3 O

Mw = 3013C x 2 15N x 1

N

N O

ON

Mw 30

Produces an ion of Mw = 115after fragmentation

NCH3

OO

Mw = 29 etc

98Produces an ion of Mw = 116after fragmentation

iTRAQ

iTRAQ

This part gives the sequence information

The low molecular weight region 114-117 contains reporter ions

100

The low molecular weight region 114 117 contains reporter ionsRatio tells us something about the relative abundance of this proteinin the 4 samples

iTRAQ reagent-8plex Protein Quantitation

Protein DimethylationProtein Dimethylation

＋ 28 Da， ‘light’

＋ 32 Da， ‘intermediate’

＋ 36 Da， ‘heavy’

Using formaldehyde globally label the N-terminus of peptide and ε-amino group of K through reductive amination.

SRM d W t Bl tSRM and Western Blots

WB SRM

Quality of the assay

based on a single antibody

depend on isotopically labeled reference peptides

Quality of the results

depends on the intensity of a band on the blot

based quantification uses multiple signals that are integrated into a composite score indicating the proteinband on the blot composite score indicating the protein quantity.

Performance characteristic

limit of detection, linear dynamic range, ability to multiplex and reproducibility For most of these characteristics MS-characteristic

sand reproducibility. For most of these characteristics MSbased methods now outperform Western blotting.

An example of SRM applicationAn example of SRM application

A set of candidate biomarkers is verified by applying SRM assays for the did t t i t f ti t d f ti t d t lcandidate proteins to fractionated sera from patient and control groups,

for example. Mutants of the target proteins can be monitored in the sampled population.

Human Proteome

2014

The adult/fetal tissues and haematopoietic cell types

Work flow

• ~25million high-resolution tandem mass 2 000 LC MS/MS• >2,000 LC-MS/MS runs

• using the MASCOT16 and SEQUEST17, P l t 18Percolator18• 293,000 non-redundant peptides (q value, 0.01 with

di t 260 ta median mass measurement error ~260 parts per billion)

Th di b f tid d di• The median number of peptides and corresponding tandem mass spectra identified per gene are 10 and 37 ti l37, respectively• the median protein sequence coverage was ~28%

Tissue-supervised hierarchicalTissue-supervised hierarchical clustering

Novel protein-coding regions in the human genomegenome

16 million MS/MS spectra that did not match currently annotated proteins

ConclusionConclusionConclusionConclusion

Proteomics is extremely valuable for understanding biological processes and g g padvancing the field of systems biology.

“The ultimate goal of systems biology is the i t ti f d t f th b tiintegration of data from these observations

into models that might, eventually, represent and simulate the physiology of the cell.”