Introduction to Proteomics

Phil CharlesCCMP

Overview of Talk

• Overview of proteomics as a concept• Techniques discussion• 2D Gels and experimental design

paradigms• Proteomics mass spectrometry• Identification• Quantitation

Proteomics is the study of the overall state of an

organism’s temporal protein composition

The biological state of the proteome is encoded in• The relative abundance of currently expressed proteins (and

their isoform)• Their localisation relative to cellular (or extracellular)

structures• Their interaction partner molecules and substrates• Their current post-translational modification state• Their folded structures• …

A Different View on Life

• Different levels of biological complexity

• More layers of regulation and control• Increased heterogeneity of samples

Genome Transcriptome Proteome … Phenotype

Why consider Proteomics?

• Orthogonal verification of gene activity.

• Observe biological state after more levels of regulation and control – closer to phenotypic outcome.

• Observe proteomes of extracellular locations – blood plasma/serum, urine etc.

Proteomics

• Classical biochemistry• Two-dimensional gels (2DGE)• Mass spectrometry• Computational analysis

Methods in Proteomics

• Separation– Gels– Immunochemistry– Chromatography

• Identification– Immunochemistry–Mass spectrometry

• Quantitation– All of the above

Identification vs Quantitation

• What’s there? How much of it is there?

• How sure are you about the ID?• How sure are you about the

abundance?• Not there versus not detectable

2DGE

• Separate proteins by isoelectric point, then by mass

• Visualise with silver staining or coomassie

• Use CyDyes to label samples so they can be run together on the same gel

Appl Microbiol Biotechnol. 2007 October; 76(6): 1223–1243.

Quantitation Experimental Paradigm - Labelling• Label samples in such a way as to not affect

subsequent processing but allow differentiation in final analysis. Examples:– Fluorescent dyes (2DGE)– SILAC amino acid labels (MS)– Isobaric mass tags (MS/ MS)

• Process multiple samples simultaneously, differentiate only in final analysis on basis of label.– Avoid some proportion of technical variance– Best to worst (for avoiding technical variance):

• Labelling in vivo• Labelling protein mixture• Labelling peptide digestion mixture

Aline Chrétien, Edouard Delaive, Marc Dieu, Catherine Demazy, Noëlle Ninane, Martine Raes, Olivier ToussaintUpregulation of annexin A2 in H2O2-induced premature senescence as evidenced by 2D-DIGE proteome analysisExperimental Gerontology, Volume 43, Issue 4, April 2008, Pages 353–359

Quantitation Experimental Paradigm – Normalising to standard• Combine each sample (labelled with

one label) with a representative standard (labelled with another label).

• Perform analysis• For each protein in each run,

normalise observed abundance in labelled sample to observed abundance in labelled standard.

Normalised Abundance Normalised Abundance Normalised Abundance

Statistical Analysis

Mass Spectrometry

• Mass Spectrometry is a technique for the detection and resolution of a sample of ions by their mass-to-charge ratio - represented by m/z where m is the mass in Daltons and z is the charge. ’

Proteomic Mass Spectrometry

• Classical biochemistry techniques and 2DGE are, in general, ‘top-down proteomics’ – identify and quantify whole proteins.

• Most modern proteomic MS is ‘bottom-up’

Shotgun/’bottom-up’ proteomics

Proteins Peptides

LNDLEEALQQAKEDLARNKLNDLEEALQQAKNVQDAIADAEQRSKEEAEALYHSKSLVGLGGTKTAAENDFVTLKTAAENDFVTLKKTSQNSELNNMQDLVEDYKTSQNSELNNMQDLVEDYKKVDLLNQEIEFLKYEELQVTVGRYLDGLTAERADLEMQIESLTEELAYLKADLEMQIESLTEELAYLKKAETECQNTEYQQLLDIK

Peptide IDs+ Quantitation

IPI:IPI00000073.2IPI:IPI00217963.3IPI:IPI00031065.1IPI:IPI00376379.4IPI:IPI00397801.4IPI:IPI00009950.1IPI:IPI00395488.2IPI:IPI00295414.7IPI:IPI00554711.3IPI:IPI00009867.3IPI:IPI00019449.1IPI:IPI00016915.1IPI:IPI00060800.5IPI:IPI00013885.1IPI:IPI00221224.6

Observed Proteins+ Quantitation

AnalysisMS-MS/

Tandem MS

SeparationSCX

High pH RP LCLow pH RP LC

SeparationSDS-PAGE

Antibody-based approaches

LNDLEEALQQACEDLAR

N KLNDLEEALQQAK

Digestion

Tandem Mass Spectrometry

Mass Analyser

+ Detector

Sample Inte

nsity

m/z

Mass Analyser

+ Detector

m/z

Inte

nsity

Mass Spectrum

Tandem Mass Spectrum MS/MS spectrum

Identification by MS/MS

• Search fragment spectrum against a database of protein sequences. For each sequence, digest into peptides, generate an expected fragment ion spectrum, and match to observed spectrum

m/z

Inte

nsity

m/z

Inte

nsity

IITHPNFNGNTLDNDIMLIK

?

Mass Analyser + Detector

Identification by MS/MS

• There are multiple commonly used MS/MS fragment spectra search engines, including:– Mascot– Sequest– OMSSA– X!Tandem– MS Amanda– Andromeda– ProteinPilot

A brief overview of Mass Spectrometric quantitation

Please feel free to stop me and ask questions!

Tandem Mass Spectrometry

Mass Analyser

+ Detector

Sample Inte

nsity

m/z

Mass Analyser

+ Detector

m/z

Inte

nsity

Mass Spectrum

Tandem Mass Spectrum MS/MS spectrum

Low pH Reverse Phase LC

‘Survey Scan’/‘MS1’/

‘MS Scan’

Select Peptide Ions

Fragmentation

CIDAlso ETD,PQD,HCD

‘Fragment Ions Scan’/‘MS2’/

‘MS/MS Scan’

time

Data-Dependent Acquisition (DDA)

Intensity

Retention Time

m/z

Intensity

Retention Time

m/z

Intensity

m/z

Intensity

m/z1/charge

(z)

Peptide Isotopomer DistributionThis is all 1 peptide

Think of it as a frequency distribution based on a probability function.

The relative intensity of each peak is the relative chance of a single peptide molecule having that m/z

Intensity

Retention Time

m/z

Intensity

m/z

Intensity

Retention Time

m/z

m/z

Intensity

IITHPNFNGNTLDNDIMLIK

Intensity

m/z

Quantitation Labelling Strategies

• MS-based strategies– In-vivo labelling (compare peak pairs) • SILAC, 15N, 18O, 2H

• MS/MS-based strategies– Isobaric Tags• iTRAQ, TMT

Intensity

Retention Time

m/z

Intensity

m/z

m/z

Intensity

Intensity

Retention Time

m/z

Intensity

m/z

m/z

Intensity

Isobaric Tag Labels e.g. iTRAQ, TMT

Intensity

Retention Time

m/z

m/z

Intensity

IITHPNFNGNTLDNDIMLIK

Intensity

m/z

Intensity

Retention Time

m/z

Intensity

m/z

m/z

Intensity

Intensity

Retention Time

m/z

Intensity

Retention Time

m/z

MS quantitation - peak pair comparison

Intensity

Retention Time

m/z

Intensity

Retention Time

m/z

Intensity

Retention Time

m/z

ID

ID

ID

ID

ID

Identification vs Quantitation

• What’s there? How much of it is there?

• How sure are you about the ID?• How sure are you about the

abundance?• Not there versus not detectable

Quantitation Software

• MaxQuant• Progenesis LC-MS• ABI Peaks• Thermo ProteomeDiscoverer• + bespoke and specific tools

The Oxford Central Proteomics Facility

• CCMP/CPF – Kessler Lab – WTCHG• CPF - Ben Thomas – Dunn School

• Computational Biology Research group - WIMM

Thank you for your attention

Please feel free to ask questions