pc219 lecture 5 30 april, 2010 [email protected] · 2010. 4. 12. · ms resolution: 60000 ms2...

1

Quantification by Mass Spectrometry

PC219

Lecture 5

30 April, 2010

[email protected]

2

Applications of Quantitative Proteomics

Detecting changes in proteomes

in space: cellular and tissue localizationin time: signaling cascades or turnoversample perturbations:

•Comparison of normal and diseased tissue samples

Biomarker discovery

•Drug development

•Diagnostics

•Gene knockdowns or over expression

•Inhibitors: eg antibodies or siRNA

•Growth factors/hormones

•Cell-cell interactions

•Drug treatment

Qualitative protein identification is NOT sufficient to describe a biological system

3

Quantitative Proteomics Methodologies

2D gel electrophoresis

silver stain

Fluorescence Difference Gel Electrophoresis

Pro-Q Diamond

2,4-dinitrophenylhydrazine

Protein expression array analysis

Global epitope tagging(Nature2003v425p737)

MS-based quantification methods

• Quantification• H/D exchange – protein surface mapping• Proteomics dynamics

4

LCMSMS

Quantification

Stable Isotope

Labeling

Label-free

methods

Metabolic

SILAC

In-vitro

iTRAQ

Survey

Intensity based

Spectral

counting

MS1

MS2MRM

Basic Classifications of MS Based Quantification

5

Label Free Quantification

6

Label Free QuantificationSpectral Counting

AnalChem2004v76p4193

•Spectral counts (# of MSMS from a Protein) correlate with protein abundance well

•Spectral counting dynamic range (single runs) is about two orders of magnitude

•10 repeated analysis can reach 95% saturation level

7

Label Free QuantificationSpectral Counting

Minimum protein ratios that can

be determined at 80 and 95%

confidence limits

MCP2005v4p1487

8

Label Free QuantificationSpectral Counting - Formula

NSAF: normalized spectral abundance factor(PNAS2006v103p18928)

SpC: total number of tandem MS spectra matching peptides from protein

L: protein length

n1, n2 - spectral counts for sample 1 and 2

t1, t2 – total spectral count (sampling depth) for samples 1 and 2

f – correction factor 0.5 (Bioinformatics2004v20pi31)

RSC: log2(protein ratio) measured from spectral counts(MCP2005v4p1487)

9

BZR1

C P

In-Vitro Phosphorylation of BZR1 by BIN2 Kinase

E. coli expressed MBP-BZR1 as substrate

E. coli expressed GST-Bin2 as kinase

Phospho sample: BZR1+Bin2+ATP

Control sample: BZR1+Bin2

ZY Wang of Carnegie Institution

Goal: Estimate Stoichiometry of PTMs

Label Free QuantificationExtracted Ion Chromatogram Based Methods

10

m/z

949.38-949.47

RT

35.0


Retention time - m/z ion map: Phospho sample (BZR1+Bin2+ATP)RT:0.00 - 60.04

0 10 20 30 40 50 60Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rela

tive A

bundance

34.95

400 600 800 1000 1200m/z

0

10

20

30

40

50

60

70

80

90

100

Rela

tive A

bundance

633.29z=3

881.89z=4

949.94z=2599.83

z=21175.52

z=3664.34z=4

750.90z=2

489.29z=2

1055.52z=2

391.29z=1

872.67z=?

1202.50z=?

Spectrum @ rt=35.0

Extracted

Ion chromatogram

for m/z range of

949.38-949.47

11


Phospho Sample Control Sample

12


160 170 180 190 200 210 220 230

1750

1800

1850

1900

1950

2000

p3

p2

p1

p0

10 20 30 40 50

p3

p2

p1p0

m/z

Retention TimeRetention Time

170 186

ISNSCPVTPPVSSPTSK

Phosphopeptides

13


259FAQQQPFSASMVPTSPTFNLVKPAPQQMSPNTAAFQEIGQSSEFK303++++

38 39 40 41 42 43 440

1

2

3

4

5

6x 104

Ret. Time (min)

*Non-constant sampling rate

*Noisy signal

*Initial peak parameters

0.9230.982R2

0.3460.319Peak Width

(min)

7.14X1032.26X104Peak Area

(min)

1.86X1045.67X104Peak Height

40.8140.85Ret. Time

(min)

PhosphoControl

m/z = 1225.63, z = 4

Extracted Ion Chromatogram and Integration

14


15 20 25 30 35 40 45 50-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

15 20 25 30 35 40 45 50-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

Retention Time (min) Retention Time (min)

Sample a

Sample b

Before After

Sample a

Sample b

LC Alignment of base-peak chromatograms:

•Dynamic Time Wrapping (similarity in chromatograms)

•Use “landmark” ions (needs MSMS info)

15

In vitro PhosphorylationEColi MBP

Control

Phosp

ho

Lo

g1

0(s

elec

ted

io

n c

hro

mat

og

ram

pea

k a

rea)

Log10(selected ion chromatogram peak area)3 3.5 4 4.5 5 5.5 6 6.5 7

3

3.5

4

4.5

5

5.5

6

6.5

7

77 peptide ion pairs

Std Dev = 11.4%

Stoichiometry of PTMs


16

3.5 4 4.5 5 5.5 6 6.5 73.5

4

4.5

5

5.5

6

6.5

7

170ISNSCPVTPPVSSPTSK186++

105VTPYSSQNQSPLSSAFQSPIPSYQVSPSSSSFPSPSR141+++

142GEPNNNM(o)SSTFFPFLR157++

259FAQQQPFSASMVPTSPTFNLVKPAPQQMSPNTAAFQEIGQSSEFK303++++

200Q(q)SMAIAK206+

207QSM(o)ASFNYPFYAVSAPASPTHR228+++

36RER38+

229HQFHTPATIPECDESDSSTVDSGHWISFQK258+++++

187NPKPLPNWESIAK199++

207QSMASFNYPFYAVSAPASPTHR228+++

229HQFHTPATIPECDESDSSTVDSGHWISFQK258++++

207Q(q)SMASFNYPFYAVSAPASPTHR228+++

158NGGIPSSLPSLR169++

200QSM(o)AIAK206++

Phosp

ho

Lo

g1

0(s

elec

ted

io

n c

hro

mat

og

ram

pea

k a

rea)

Control

Log10(selected ion chromatogram peak area)



In vitro Phosphorylation: BZR1_ARATH

17

User Interface

- Scatter Plot, Extracted IC, Peptide ID Page


18

BRASSINAZOLE-RESISTANT 1 protein (BZR1)

Extent of Phosphorylation

S, T, and Y are conformed assignments

in this experiment

S and Y are tentative assignment

10 20 30 40 50

60 70 80 90 100

110 120 130 140 150

160 170 180 190 200

210 220 230 240 250

260 270 280 290 300

310 320 330

---------- ---------- -RRKPSWRER ENNRRRERRR RAVAAKIYTG

LRAQGDYNLP KHCDNNEVLK ALCVEAGWVV EEDGTTYRKG CKPLPGEIAG

TSSRVTPYSS QNQSPLSSAF QSPIPSYQVS PSSSSFPSPS RGEPNNNMSS

TFFPFLRNGG IPSSLPSLRI SNSCPVTPPV SSPTSKNPKP LPNWESIAKQ

SMAIAKQSMA SFNYPFYAVS APASPTHRHQ FHTPATIPEC DESDSSTVDS

GHWISFQKFA QQQPFSASMV PTSPTFNLVK PAPQQMSPNT AAFQEIGQSS

EFKFENSQVK PWEGERIHDV GMEDLELTLG NGKARG

<10%

~85%

~90%

~80%

~45% ~30%

~70%

<10% <10%


19


Data Processing Workflow

.Raw File

Survey Scans

MS2 Scans

Peak Extract

IDed

Ion Chrom.

Quantitation

Results

Peptide/

Protein ID

DataBase

SearchRun Comparison

Integration

20


JProteomeRes2008v7p51

Proteomics2008v8p731

21

Label Free QuantificationExtracted Ion Chromatogram Based Method - OpenMS

BMCBioinformatics2008v9p163

Isotope distribution modeling: wavelet

Software architecture: C++/Linux

22

Label Free QuantificationExtracted Ion Chromatogram Based Method

Experimental

Consistency in sample collection and processing

LC and MS stability

Replicate analysis

Data Prcessing

•Data format

•Data visualization

•Normalization

Mass recalibration

LC retention time alignment

Abundance normalization

•Data quality assessment

•Result analysis

Difference detection

Multivariate analysis

Log(Peptide abundance)Log(R

eplic

ate

peptid

e r

atio)


23

Stable Isotope Labeling

24

Experimental StrategiesIntroduction of Controls in Stable Isotope Labeling

(QconCAT:Spiked Q-proteins)

Proteomics2008v8p4873

25

Absolute Quantification (AQUA) with Internal Standard Peptides

PNAS2003v100p6940

26

QconCATTryptic standard peptides from artificial proteins

NatMethods2005v2p587

Artificial protein

Containing

concatenated tryptic Q peptides

and his-tag

27

Stable Isotope Chemical Labeling

28

stable isotope chemical labelingdimethyl labeling of ε-amine

NatProtocol2009v4p484

•Can be performed (a) in-solution, (b) online with LC-MS or (c) on-column(SepPak)

•2 carbons and 6 hydrogens for labeling – in principle

•Quantification in MS or survey scan level

29

stable isotope chemical labelingMethyl esterification of carboxyl groups

•Perform in dry condition (sample, glassware, etc)

if a peptide has 10 Es or Ds and single site labeling efficiency is 99%,

only 90% of peptide is fully labeled

•Help to increase charge states (better sensitivity)

decrease acidic groups (better fragmentation)

improve phosphopeptide enrichment

•Quantification in MS or survey scan level


C

O

OH

+ CH3OH C

O

OCH3

+ H2O

C

O

OH

+ CD3OH C

O

OCD3

+ H2O

30

Isotope coded affinity tag (ICAT)

CurOpinionBiotechnol2000v11p396

•Purify peptides by streptavidin beads

•Only labels cysteines

•The tag is too large for MS

•Difficult to elude peptides

31

Isotope coded affinity tag (ICAT)

http://www.absciex.com/LITERATURE/cms_040324.pdf

•Acid cleavable linker facilitates release•Heavy and light carbon allows for co-eluting peptides•The structure is not disclosed

Early usage: AnalChem2006v78p4543

32

metal-coded affinity tag (MeCAT)

MCP2007v6p1907

thiol-specific labelingspacermetal coding

•Labeling group: cysteines

•Metals used: 159Tb 165Ho 169Tm and 175Lu

•Detection limit: 100amole

33

Isobaric Tags for Relative and Absolute Quantitation (iTRAQ)

MolCelProteomics2004v3p1154

34

Isobaric Tags for Relative and Absolute Quantitation (iTRAQ)

MolCelProteomics2008v7p853

35

Tandem Mass Tag (TMT)

http://www.piercenet.com/Objects/View.cfm?type=Page&ID=B60171CD-5056-8A76-4E36-958F5C186495#tmtstruct

6 Channels

36

MS Instrumentation for iTRAQ

iTRAQ was initially introduced on Q-TOF type instruments

•MS3 in ion traps(http://www.thermo.com/eThermo/CMA/PDFs/Various/File_27402.pdf)

•PQD for detecting fragment ions on ion traps(MCP2008v7p1702)

•HCD for detection on Orbitraps

LTQ Velos Oritrap

•ETD (or combined with CID) can

generate reporter ions(AnalChem2009v81p1693)

m/z

101-108m/z

113-121

“1/3 Rule”

37Jonathan Trinidad

iTRAQBoth quantification and identification info is contained in MSMS

Reporter Ions

Peptide fragment ions contain isobaric tags

38

Quantification with TMT

Mass Spectrometer: Thermo Scientific LTQ Orbitrap VelosMS Resolution: 60000

MS2 Resolution: 7500

MS AGC target: 1e6

MS/MS AGC target: 5e4

Exclusion mass tolerance: 10 ppmInjection Time FTMS/MS: 200 ms

Full MS mass range: 400–1400 m/z

MS/MS Mass range: 100–2000 m/z

Isolation 1.2 Da

MS/MS Events: Full MS in Orbitrap followed by top ten data dependent HCD MS/MS

CE for HCD: 45%

Terry Zhang

E. Coli digestQuantifiable peptides

•74% (40ng)

•81% (80ng)

Quantification Errors

•20% (40ng)

•15% (80ng)

39NatBiotech2007v25p994

iTRAQ

Quantification of Phosphorylation Levels

Kinobeads: ~1000 kinase inhibitors(http://www.cellzome.com/files/kinobeads.pdf)

40

iTRAQ

Phosphorylation Levels and Signaling Pathways

PNAS2007v104p12867

EGFR pathways: Different receptor level in U87MG glioblastoma cell lines

41

iTRAQlocalization of organelle proteins by isotope tagging (LOPIT)

NatProtocols2006v1p1778

vacuolar membrane

ER

PM

Golgi apparatus

mitochondria_plastids

Principal component analysis (PCA)

Reduction of dimensions

42

Quantification

Data Analysis – Clustering Analysis

PNAS2007v104p12867

Self Organized Maps (SOM)

MCP2008v7p684

Hierarchical Clustering

Reference: PNAS1998v95p14863

Freeware: http://rana.lbl.gov/EisenSoftware.htm

43

Proteolytic 18O Labeling

Electrophoresis1996v17p945

Trypsin introduces a mixture of

one and two 18O incorporated

peptides

LysN incorporates a single 18O at

high pH


MCP2005v4p1550


44

Metabolic Stable Isotope Labeling

MCP2010v9p11

MCP2002v1p376

PNAS1999v96p6591

Phytochemistry2004v65p1507

PlantJ2008v56p840

45

Metabolic Stable Isotope LabelingStable Isotope Labeling by Amino Acids in Cell Culture (SILAC)

NatBiotechnol2004v22p1139

46

Multiple Reaction Monitoring(MRM)

Term coined by Cooks et al in 1978(AnalChem1978v50p2017)

Used by pharmaceutical industry for years

Drug metabolites analysis

Performance improved by introduction of Qtrap instruments(DDTtarget2004v3pS31)

Modern instruments have MRM dwell time of 5msec

Sensitivity of <1fmole

47

Multiple Reaction Monitoring(MRM)information-dependent acquisition (IDA) to MRM Transitions

PNAS2007v104p5860

48

Multiple Reaction Monitoring(MRM)Specificity of MRM Transitions – Fragment Ion Coelution

PNAS2007v104p5860

49

TM

Immature MatureSU

Gag

MA

CA

RNANC

MA CA p2 NC p1 p6MA CA p2 NC p1 p6

Gag (55 kDa)

24 kDa

HIV

Hydrogen/Deuterium ExchangeStudy of High Order Protein Structures

A.G. Marshall

50

Protein or Complex

D

D

D

D

D

D

D

H

H

Dilute 10 fold

w/ D2O

buffer

H/D exchange

Quench

Digest with pepsin in ice water for 3 min

pD 2.5 ~ 3.0

Freeze in

liquid N2

& store

at -70 oC

Thaw

on ice

InjectMicro-bore c8 column

30 µµµµL/min

SplitterSplitterSplitterSplitter

to wasteto wasteto wasteto waste

Online LC/FT-ICR MS

Ice water

Ice water


A.G. Marshall

51

PIVQNLQGQM VHQAISPRTL NAWVKVVEEK AFSPEVIPMF SALSEGATPQ

DLNTMLNTVG GHQAAMQMLK ETINEEAAEW DRLHPVHAGP IAPGQMREPR

GSDIAGTTST LQEQIGWMTH NPPIPVGEIY KRWIILGLNK IVRMYSPTSI

LDIRQGPKEP FRDYVDRFYK TLRAEQASQE VKNWMTETLL VQNANPDCKT

ILKALGPGAT LEEMMTACQG VGGPGHKARV L

Peptic Peptides Cover 95% of CA Primary AA Sequence

1

51

101

151

201 231

A.G. Marshall


52

H/D Exchange

Period (min)

4080

0

0.5

2

60

240

843 846 849m/z

Assembled CA Monomer CA

Y169-L189

A.G. Marshall


53

1 10 100 1000

1490

1491

1492

1493

1494

1495

1496

1497

1498

Time (min)

Bottom of Helix III Protected upon AssemblyCentroidMass CA

Assembled CA

A.G. Marshall


54

1 10 100 10001934

1935

1936

1937

1938

1939

1940

1941

Time (min)

CA

Assembled CA

Helices VI and VII Not Protected upon AssemblyCentroidMass

A.G. Marshall


55



H/D by Topdown Analysis

Fully deuterated protein

+1H2O/CH3CO2N

1H4 at pH 3.5

56

Dynamic ProteomicsStudy Protein Turnover on A Proteomic Scale

FunctionalGenomicsProteomics2005v3p382

57

Dynamic ProteomicsExperimental Strategies


AnalChem2004v76p86

Dynamic SILAC

Synthesis/Degradation Ratio

Mass Spectrometry

58

Dynamic ProteomicsDynamic SILAC

FunctionalGenomicsProteomics2005v3p382

Chicken fed with stable isotope labeled valine

VKVGVNGFGR

Re

lative

Iso

top

e A

bunda

nce

59

Dynamic ProteomicsSILAC Mouse

Cell2008v134p353

60MCP2009v8p2653

Dynamic ProteomicsWater Labeling

incorporation of 2H or 18O into plasma albumin

by bolus injection

61

Conclusions

•Label free quantification was originated and is still driven by biomarker discovery

•Absolute quantification is possible with spiked-in or QconCAT calibrant peptides

•SILAC allows mixing at protein level

•iTRAQ has been widely used for studying signaling pathways

•Dynamics of a proteome can be probed by metabolic labeling

pc219 lecture 5 30 april, 2010 [email protected] · 2010. 4. 12. · ms resolution: 60000 ms2...

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