Mass Spectrometry based Proteomics and Metabolomics - PowerPoint Presentation

Slide1

Mass Spectrometry based Proteomics and Metabolomics

Guoan ZhangDirector Proteomics and Metabolomics Core Facility

Proteomics & Metabolomics

Core Facility

@

WCM

Slide2

Proteomics & Metabolomics Core Facility

@Weill Cornell MS- based protein/metabolite

analysis

Opened for services

Oct 2016 for proteomicsMay 2017 for metabolomics

Proteomics & Metabolomics

Core

Facility

@

WCM

Slide3

The rise of OMICS according to Pubmed

Proteomics & Metabolomics Core

Facility

@

WCM

Proteins and metabolites are more difficult to study because they are more dynamic and diverse

Slide4

Coverage comparison: genomics > proteomics >> metabolomics

Genomics100% coverageProteomics

Can identify ~10,000 protein

vs

~20,000 genes (human)Low sequence coverageMetabolomicsCan detect ~1000 metabolites

vs

? total metabolites

Needs multiple platforms

Proteomics & Metabolomics

Core

Facility

@

WCM

Slide5

Mass spectrometry

Proteomics & Metabolomics Core

Facility

@

WCM

Slide6

Ion source makes a molecule charged

and transfers it to gas phase

Proteomics & Metabolomics

Core

Facility

@

WCM

Slide7

Matrix-assisted laser desorption ionization (MALDI)

Proteomics & Metabolomics

Core

Facility

@

WCM

Slide8

Electrospray ionization

Proteomics & Metabolomics Core

Facility

@

WCM

Mass Spectrometry Reviews 20(6):362-87

Slide9

Analyzer

sorts the ions based on their mass-to-charge ratio

Proteomics & Metabolomics

Core

Facility

@

WCM

Slide10

Ion trap

Proteomics & Metabolomics Core

Facility

@

WCM

http://

www.shimadzu.com

Slide11

Quadrupole

Proteomics & Metabolomics

Core

Facility

@

WCM

http://

www.shimadzu.com

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Time of flight (TOF)

Proteomics & Metabolomics Core

Facility

@

WCM

http://

www.shimadzu.com

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Orbitrap

Proteomics & Metabolomics Core

Facility

@

WCM

https://

en.wikipedia.org

/wiki/

Orbitrap

Slide14

MS1, MS2 (MS/MS), and MSn

Proteomics & Metabolomics

Core

Facility

@

WCM

http://

planetorbitrap.com

Slide15

Fusion Lumos

OrbitrapProteomics & Metabolomics

Core

Facility

@

WCM

http://

planetorbitrap.com

Slide16

Q Exactive

OrbitrapProteomics & Metabolomics

Core

Facility

@

WCM

http://

planetorbitrap.com

Slide17

Proteomics & Metabolomics

Core Facility @

WCM

https://

en.wikipedia.org

Slide18

shotgun proteomics (bottom up)

Nature Immunology 15, 112–117 (2014)

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Proteomics Services

Protein IdentificationID of proteins from SDS-PAGE and of proteins in-solution. Post-Translational Modification Analysis

Phosphorylation;

ubiquitination

; acetylation; other PTMs. Protein QuantitationLabel

-free (intensity based / spectral counting);

stable

isotope labeling by amino acids in cell culture (SILAC);

tandem mass tags (TMT);

spiked

standards.

MS

Data AnalysisProteomics & Metabolomics Core

Facility

@

WCM

Slide20

PTM analysis: affinity

enrichment is often required (peptide level)Phosphorylation (pSer

,

pThr

, pTyr):TiO2IMAC

pTyr

:

pTyr antibodyacetylation:

Ac-antibodyUbiquitination

diGly

antibody

Proteomics & Metabolomics

Core Facility

@

WCM

Slide21

Enrichment of ubiquitination by di-glycine antibody

Proteomics & Metabolomics

Core

Facility

@

WCM

Slide22

Quantitation methods

SILAC (Stable Isotope Labeling with Amino acids in Cell culture)TMT (tandem mass tag)

Label free

Proteomics & Metabolomics

Core

Facility

@

WCM

Slide23

SILAC

(S

table

I

sotope L

abeling with

A

mino acids in

C

ell culture

) can compare up to 3 samples

Picture from

wikipedia

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R

XXXXXXXX

R

Slide24

TMT (tandem mass tag

) quantitation:chemical labeling of up to 10 samples

Proteomics & Metabolomics

Core

Facility

@

WCM

Slide25

Label free quantitation:

can compare unlimited number of sample but accuracy not as good as labeled methodsProteomics & Metabolomics

Core

Facility

@

WCM

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Different protein quantitation methods have different levels of accuracy

26

Slide27

Comparison of different Quantitation methods

Proteomics & Metabolomics

Core

Facility

@

WCM

Slide28

Identification of Protein-protein interactions by MS

Proteomics & Metabolomics

Core

Facility

@

WCM

Slide29

Quantitation is almost always needed to find true

interactors from non-specific bindersProteomics & Metabolomics

Core

Facility

@

WCM

~1000 proteins identified

3 of them are specific

Slide30

APEX for identification of

transient or weaker protein binders

Proteomics & Metabolomics

Core

Facility

@

WCM

Nature

Protocols

11, 456–475 (2016)

APEX2

(ascorbate peroxidase) generates

biotin-

phenoxyl

radicals that covalently tag proximal endogenous proteins

Slide31

Site specific quantitation of phosphorylation changes upon treatment of primary neurons

Medium

Heavy

4 biological replicates (2 forward/reverse)

Proteomics & Metabolomics

Core

Facility

@

WCM

Slide32

Complete SILAC labeling of cells that divide is straightforward

5 divisions

Isotope incorporation

>

97

% for zero turnover proteins after 5 cell divisions

Light medium

Heavy medium

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Complete SILAC labeling of post-mitotic cells is difficult

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Sample A, Protein X

(Light: Lys0/Arg0)

Sample B, Protein X

(Heavy: Lys6/Arg6)

m/z

%

0

100

m/z

%

0

100

m/z

%

0

100

m/z

%

0

100

Incomplete labeling changes SILAC ratios

Slide35

Sample A, Protein X

(Medium: Lys4/Arg6)

Sample B, Protein X

(Heavy: Lys8/Arg10)

m/z

%

0

100

m/z

%

0

100

m/z

%

0

100

m/z

%

0

100

Multiplex labeling allows SILAC quantitation with partially labeled cells

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Forward 1

24119

Forward 2

25844

Reverse 1

22561

Reverse 2

25746

2133

2680

1658

2455

1560

14164

2058

1173

2870

873

628

1398

661

1319

839

High overlap of

phosphosite

identification from four biological replicates

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SILAC ratio correlation between biological replicates

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Many phosphosites

are regulated by stimulation

> 20,000 sites

were quantified

2 fold change:

355 up; 17 down;

267 proteins

FDR 1.7 %

1.5 fold change:

708 up; 98 down;

520 proteins

FDR 4.4%

Proteomics & Metabolomics

Core

Facility

@

WCM

Slide39

Changing sites revealed interaction networks

transcription

Cytoskeleton

rearrangement

Proteomics & Metabolomics

Core

Facility

@

WCM

Slide40

Metabolomics

Proteomics & Metabolomics Core

Facility

@

WCM

Slide41

Many analytical platforms are used for metabolomics

NMRGC/MSLC/MSCE/MSLC/MS has the best coverage and dynamic range

Slide42

Untargeted metabolite profiling

Proteomics & Metabolomics Core

Facility

@

WCM

Attempts to identify and quantify all compounds

Needs standard MS/MS library

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Targeted polar metabolite profiling

Proteomics & Metabolomics Core

Facility

@

WCM

80% methanol

HILIC

(hydrophilic interaction chromatography)

A list of >200 compounds with known m/z and RT

Slide44

Triple

quadrupole

vs

Orbitrap

for targeted

metabolimics

Proteomics & Metabolomics

Core

Facility

@

WCM

Only acquires signals of interest

Acquires all signals and filter later to obtain signals of interest

Slide45

High sample prep reproducibility is essential as metabolites are highly dynamic

Proteomics & Metabolomics Core

Facility

@

WCM

Slide46

PCA analysis to illustrate separation of different experimental groups

Proteomics & Metabolomics Core

Facility

@

WCM

Slide47

Clustering of different experimental groups

Proteomics & Metabolomics Core

Facility

@

WCM

Slide48

Isotope tracing

Proteomics & Metabolomics Core

Facility

@

WCM

GENETICS July 1, 2017 vol. 206 no. 3 1169-1185

Slide49

High resolution MS

enables isotope tracing analysis by comparing unlabeled and C13 labeled isotopologuesProteomics & Metabolomics

Core

Facility

@

WCM

C12 sample

C13 sample

Example

: Glutamate (C5H9NO4

), all 5 possible

isotopologues

are compared

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Profiling of C13 labeled polar metabolites

Proteomics & Metabolomics Core

Facility

@

WCM

Slide51

We are in the process of establishing LC-MS based

lipidomics profiling assaysProteomics & Metabolomics

Core

Facility

@

WCM

Slide52

Contact us

Websitehttps://wcmc.corefacilities.org/service_center/show_external/3474

Director

Guoan

Zhang, Ph.D.Office: BRB-1616Tel: (646) 962-6222

Email

: guz2004

@med.cornell.eduLab

BRB-1552Tel:646-962-9739

Proteomics & Metabolomics

Core

Facility

@

WCM