Introduction to Proteomics

Room 4508, Level 5, Microbiol-Genetics Building

Department of Genetics, Faculty of Science, KU

Dr. Teerasak E-kobon

Outline

1. Proteomic basics

2. Mass spec-based proteomic workflow

3. Protein identification by MS, MS/MS

4. Protein quantification by MS

5. Summary

What is Proteomics ?

“ Analysis of the complete complements of proteins

Proteomics includes not only the identification and

quantification of proteins, but also the determination

of their localization, modifications, interactions,

Activities, and ultimately, their functions.”

Stan Fields (Science, 2001)

Or, the proteins present in one sample at a certain point in time.

Amino

Acid

Properties

Direct protein sequencing

Pevsner, Jonathan. (2015). Bioinformatics and Functional Genomics. 3rd Eds. Wiley, UK.

- Edman degradation process is

illustrated for a protein

fragment of six amino acids.

The first amino acid reacts

through its amino terminus

with phenylisothiocyanate

(PITC).

- Under acidic conditions this

amino acid residue, derivitized

with phenylthiohydantoin

(PTH), is cleaved and can be

identified in an amino acid

analyzer.

- The peptide now has five

amino acid residues, and the

cycle is repeated with

successive amino-terminal

amino acids.

What kind of answers can we get?

Pevsner, 2015

Conceptual proteomics experiments

Pevsner, 2015

Proteomics workflow

-1D/2D gel

-Column chromato-

graphy

-Immunoprecipitation

-Pulldowns with

tagged proteins

-Affinity depletion

Cell culture

Model organism

Body fluids

Tissues

- RP-HPLC

- Strong cation exchange (SCX)

- Weak anion exchange (WAX)

- Hydrophilic interaction (HILIC)

- Metal affinity (IMAC)- Peptide ionization (ESI, MALDI)

- Peptide fragmentation (CID, HCD, ETD, ECD, IRMPD, ISF)

- Mass analyzer (Ion trap, time-of-flight, quadrupole, orbitrap/ICR)

- Database search

- Acurate mass

time tag

- Peptide mass

fingerprinting

- Metabolic labeling

(SILAC)

- Chemical protein

/peptide (iTRAQ,

TMT)

- Label free

(spectral count,

extr. Ion chrom)

- Multiple reaction

monitoring (MRM)

Bottom up VS Top down Proteomics

“Bottom up” proteomics

Sample

- cells

- Body fluids

- Tissues

- etc

Protein

extraction

- Whole cell

lysate

- Protein

purification

Peptides

- Separation

- Clean up

Mass spectrometry

- Untargeted

analysis

- Targeted analysis

Data analysis

- Database

search

- TPP

- Quantification

“Top down” proteomics Pevsner, 2015

Sample Preparation

1. Extract and separate proteins

2. Digest proteins

3. Separate and clean up peptides

-no (low) detergent & low/no salt

Protein Digestion

hh

https://www.thermofisher.com/in/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-

protein-methods/sample-preparation-mass-spectrometry/_jcr_content/MainParsys/image_b5be.img.jpg/1437659928778.jpg, 9 Jan 2016

Protein separation: 1-DE

https://upload.wikimedia.org/wikipedia/commons/4/46/SDS-PAGE_Electrophoresis.png; http://static-

content.springer.com/esm/art%3A10.1186%2F1477-5956-7-32/MediaObjects/12953_2009_136_MOESM5_ESM.jpeg, 9 Jan 2016

Protein separation: 2-DE

1st dimension: Isoelectric focusing (charge)

2nd dimension: SDS-PAGE (mass)

http://biochimej.univ-angers.fr/Page2/COURS/9ModulGenFoncVeg/6Proteomique/3Figures/1FigGenerales/2PrincipeGel2D.gif, 9 Jan 2016

Different gel staining methods

Sensitivity Dynamic range MS-compatible

Coomassie 8 ng 10-30 x yes

Silver 1 ng <10x Not without

special

precautions

Fluorescent 2 ng 3 orders of yes

magnitude(ProQ Emerald

Sypro Ruby)

Comparative 2-DE gels image analysis

Zucchi et al., 2001

ExPASy 2-DE gel database

Pevsner, 2015

Differential expression by DIGE

Internal

standard

Protein

extract 1

Label with Cy3

Protein

extract 1

Label with Cy5

Mixed labelled extracts2-DE

separation

Variable mode

Imager

Differential

Analysis

software

- 2D Differential Gel Electrophoresis

- Labelled N-terminus lysines with fluorescent dyes

Pevsner, 2015

How much proteins do I need?

ng of total

proteins

ug of total

proteins

mg of total

proteins

10s-100s of protein ID’s

100s-1000s of protein ID’s

1000s-10000s of protein ID’s

Proteome wide PTM

From Proteins to Peptides

NH2+ RCOOH+

NH2+ KCOOH+

NH2+

RCOOH+

Enzymatic

digest

Detectable size range

~ 8-25 amino acids or m/z <2000

… R.AR.ESAMPLER.SPEPTIDE

Trypsin cleaves C-terminal of

Lys and Arg.

Less common enzymes:

ArgC, LysC, Pepsin, etc

Trypsin generates + charged residue on C-terminal of

peptide which facilitates y-ion production in MS/MS.

Westermeier R., Naven, T., Hopker, H.R. 2008. Proteomics in Practice. Wiley-Blackwell, US. pp. 502.

Other Proteases

Westermeier et al., 2008

Mass Spectrometry (MS)

Main components:

1. Ionization

2. Mass analyzer

3. Detector

= a scale measuring the mass of a charged

molecule, mass to charge ratio (m/z)

MS or MS/MS

http://www.sec.psu.ac.th/web-board/content/view_img.php?id=5282, 9 Jan 2016

Ionization: Electrospray Ionization

Solvent evaporation

Coulomb explosionCharged peptides

In gas phase

(M + nH)n+

Multiple charged ions

https://encrypted-tbn2.gstatic.com/images?q=tbn:ANd9GcQ2LynTDoVWWHb2W6eg7mBcouozeVlMUuCvwl0n4D7aXoIgHW1i9g, 9 Jan 2016

Matrix-assisted

Laser Desorption/

ionization

Ionization: MALDI

https://upload.wikimedia.org/wikipedia/commons/thumb/a/a9/Maldi.svg/1280px-Maldi.svg.png, 9 Jan 2016

MALDI Mass Spectrum

Westermeier et al., 2008

Mass Analyzer: TOF, TOF/TOF

Linear time-of-flight MS

Reflector time-of-flight MS

- Mass range up to 350 kDa

- High sensitivity

- Low resolution

- Mass range up to 5000 kDa

- Low sensitivity

- High resolution

Westermeier et al., 2008

Mass Analyzer: Quadrupole

- Mass range: < 3000 m/z

- Resolution: up to ~2000

- Accuracy: 0.1%http://www.chemicool.com/img1/graphics/quad-sch.gif, 9 Jan 2016

Mass Analyzer: Ion trap

- Mass range: typical < 2000 m/z, extended <4000m/z

- Resolution: up to ~10000

- Accuracy: 0.1-0.01 %https://encrypted-tbn3.gstatic.com/images?q=tbn:ANd9GcQaExNWDiKMtyTzjhW2bl5yCtIDjnijXXnks1w83tXpdLA9tBb4, 9 Jan 2016

Mass Analyzer: FT-ICR

- Mass range: typical < 2000 m/z, extended <4000m/z

- Resolution: up to ~1,000,000

- Accuracy: < 5 ppm

FT-ICR (Fourier Transform Ion Cyclotron Resonance)

http://people.whitman.edu/~dunnivfm/C_MS_Ebook/CH5/Figures/Fig_5_21_e_Overview.jpg, 9 Jan 2016

Mass Analyzer: Orbitrap

- Mass range: typical < 2000 m/z, extended <6000m/z

- Resolution: up to ~240,000

- Accuracy: < 2 ppmhttp://media.americanlaboratory.com/m/20/article/18815-fig2.jpg, 9 Jan 2016

Different Mass Spectrometers

- MALDI-TOF MS

- MALDI-TOF/TOF MS/MS

- nanoLC-ESI Q/TOF MS

- Orbitrap

- MudPIT

(Multidimensional Protein

Identification Technology)

http://www.sec.psu.ac.th/web-board/content/view_img.php?id=5282, 9 Jan 2016

MudPIT

https://tanlab.files.wordpress.com/2008/10/mudpit-workflow.jpg, 9 Jan 2016

Proteomic data formats1. Mass spec data files

- Xcalibur (.raw)/Thermofinnigan

- Analyst (.wif;.t2d)/ Life Technologies

- Masslymx (.raw)/ Waters

- .baf/ Bruker

2. Analyzed data file formats

- .dta .out / Sequest

- .t2d .group / ProteinPilot

- .xml / X! tandem

- .xml .omx / OMSSA

- .mgf .dat / Mascot

- .mzxml, .pepxml, .mzData, .protxml, .mzml

*** The Human Proteome Organization (HUPO) supports a Proteomics

Standards Initiative (PSI) with the goals of defining standards for

proteomic data representation to facilitate the comparison, exchange,

and verification of data.

Protein identification by MS

(1) Peptide mass fingerprint

or peptide mass map

(2) MS/MS based peptide ID

or tandem MS

Westermeier et al., 2008

Calculating mass of a peptide or protein from m/z

m/z = (M + 1H)/z = 1233.56 [z=1]

(M + 1H) = (1233.56)*(z)

= (1233.56)*(1)

= 1233.56

M = 1233.56 – H

= 1233.56 – 1.0073

= ~ 1232.55 Da

* For MALDI-MS *Westermeier et al., 2008

Calculating mass of a peptide or protein from m/z

m/z = (M + 2H)/z = 617.28 [z=2]

(M + 2H) = (617.28)*(z)

= (617.28)*(2)

= 1234.56

M = 1234.56 – 2H

= 1234.56 – 2*(1.0073)

= ~ 1232.55 Da

* For ESI-MS *

Westermeier et al., 2008

Determination of charge state and MW

Charge state peaks

Isotope peaks

n = charge number

n = 1/Δ(m/z)

MW = (m/z)*z - nH

1

2

Solve sets of two equations

m/z = (MW + n)/n 1

m/z = (MW + n + 1)/(n + 1) 2 Westermeier et al., 2008

Peptide Mass Fingerprint (PMF)

Each peak = m/z of the peptide ionWestermeier et al., 2008

Mass Error

Mass error (ppm) = MS mass measurement error

= error/mass

Ex. A peak of mass 1529.7348, with accuracy of 10 ppm

10*10-6 = (mass error)/(mass of peak)

1*10-6 = (mass error)/(1529.7348)

mass error = (1529.7348) * (1*10-5) = 0.0153

Therefore, peak mass = 1529.7348 + 0.0153

Mass Accuracy

True mass = 1529.7348

Measured mass = 1529.7501

Δmass = 0.0153

Mass accuracy = (0.0153/1529.7348)*106

= 10 ppm

Mass Tolerance= the error window on experimental peptide

mass values

Mass tolerance = Mass Error(accuracy) * Peak Mass

= 65 ppm * 1529

= 0.1

MS/MS or Tandem MS

Select a peptide ion

Fragment a peptide ion

Measure fragment ion

mass spectra (MS/MS spectra)

Known as product ions

- To sequence individual peptides

(de novo protein sequencing)

Westermeier et al., 2008

Peptide Fragmentation in MS/MS

Collision induced

dissociation

CID

Peptide Identification

b1

b2

b3

b4

b5

b6

b7

b8

b9

y9

y8

y7

y6

y5

y4

y3

y2

y1b-ion contains N-terminus.

y-ion contains C-terminus.

Superscripted number = numbers of amino acids

Westermeier et al., 2008

Peptide Fragmentation in MS/MS

CID Spectra (Tandem MS)

y3

m/z

Rela

tive inte

nsi

ty

y4

y5

y6

y7

y8

y9

b3

b5

b6

b7

b8

Westermeier et al., 2008

Protein Coverage

Beck et al., 2001

They identified > 10,000 proteins, 174,066 peptides.

In complex mixtures many low abundance proteins

will be identified by only a single unique peptide.

Protein coverage depends on:

- Complexity of mixture

- Protein amount

- Instrument settings

- Peptide length/enzyme used

De novo sequencing of unknown peptides

Millares et al., 2012

They identified > 10,000 proteins, 174,066 peptides.

Mass changes associated with PTM(Post-translational modification)

Westermeier et al., 2008

IMAC (Immobilized-metal affinity chromatography)

For phosphorylation

Westermeier et al., 2008

Protein-Protein Interaction

Co-Immunoprecipitation (Co-IP)

Westermeier et al., 2008

Quantitative Proteomics

To quantify we need an internal or external standard with

identical physiochemical properties.

Internal standards = stable isotopic labelling

External standards = measuring the same peptide in

two consecutive runs

SILAC iTRAC/TMT AQUA

Label free quantification Targeted quant MRM AQUA

Westermeier et al., 2008

SILACStable isotope labelling by amino acids in cell culture

Metabolic labelling

Westermeier et al., 2008

SILAC

Westermeier et al., 2008

Isotopic LabellingiTRAQ (isobaric tags for relative and absolute quantification)

http://image.slidesharecdn.com/quantitativeproteomics-130430024445-

phpapp01/95/quantitative-proteomics-9-638.jpg?cb=1367289927, 9 Jan 2016

iTRAQ workflow

Fragmentation produces reporter ions

from m/z 114, 115, 116 and 117.

Westermeier et al., 2008

iTRAQ workflow

Westermeier et al., 2008

Tandem Mass Tag (TMT)

Isotopic labelling

Pros: Applicable to all samples

Relatively easy

Multiplexing (up to 8)

Cons: Cost

Separate sample processing

Westermeier et al., 2008

Other Isotopic Labellings- ICAT (do/d8) and ICAT 13C0/

13C8

(Isotope-coded affinity tags, Cys-containing proteins)

- do/d10 propionic

anhydride

(N-terminal labelling)

- 15N/14N

(whole cell labelling)

- 18O/16O (by trypsin)

Westermeier et al., 2008

Label free quantificationRelative quantification

- Extract ion

chromatogram

- Spectral counting

Pros: Cons:

- Applicable to all sample types - Susceptible to

- Cheap technical variation

- Multiplexing (infinite)Westermeier et al., 2008

Targeted QuantificationDiscovery MS/MS mode

Targeted MRM (multiple reaction monitoring) or

SRM (selected reaction monitoring) mode

Random precursor

m/z selection

Precursor m/z

fragmentation

All fragment

m/z analysis

Database search

Peptide IDs

Targeted precursor

m/z isolation

Precursor m/z

fragmentation

Selected fragment

m/z analysis

Quantification

Westermeier et al., 2008

Targeted Quantification/Identification

Known peptide

precursor m/z Known fragment m/zWestermeier et al., 2008

Targeted Quantification

Westermeier et al., 2008

PRIDE

PRoteomics IDEntifications

(PRIDE) database at the

European Bioinformatics

Institute website.

PRIDE is a central public

repository for mass

spectrometry-based

proteomics data.

Summary- Proteomics is the large-scale study of proteins, particularly

their structures and functions.

- Protein separation (electrophoresis and liquid

chromatography) and mass spectrometry (MS and MS/MS)

are important keys to protein identification and

quantification.

- Proteomics approaches can be discovery- and targeted-

modes.

- Bioinformatics facilitate high throughput proteomics data

analyses and management.

References

• Pevsner, Jonathan. (2015). Bioinformatics and Functional Genomics. 3rd Eds.

Wiley, UK.

• Westermeier R., Naven, T., Hopker, H.R. 2008. Proteomics in Practice. Wiley-

Blackwell, US. pp. 502.