Introduction to Proteomics CSC8309 - Gene Expression and Proteomics Simon Cockell Bioinformatics Support Unit Feb 2008.

Introduction to Proteomics CSC8309 - Gene Expression and

Proteomics

Simon CockellBioinformatics Support Unit

Feb 2008

Outline

• Introduction– Why proteomics?

• Sample Collection• Separation Techniques

– Gels– Columns

• Mass Spectrometry– Ionisation– Mass Analysis– Protein Identification

The proteome

• Organisms have one genome

• But multiple proteomes

• Proteomics is the study of the full complement of proteins at a given time

Why proteomics?

• Microarrays are easier, and more established– So why use proteomics at all?

• It is proteins, not genes or mRNA, that are the functional agents of the genome

• Transcriptome information is only loosely related to protein levels– Abundant transcripts might be poorly

translated, or quickly degraded

Basic principles

• 3 steps to most proteomics experiments– Preparation of a complex protein

mixture– Separation of protein mixture– Charaterisation of proteins within

mixture

Sample Collection

• Controlled conditions• Low-salt (for later Mass Spec)• Prevention of:

– Contamination– Degredation

• Consider difficult to purify proteins– e.g. membrane-bound

Separation Techniques2D Gel Electrophoresis

Separation Techniques2D-GE - Isoelectric Focusing

• Separation of proteins on basis of isoelectric point

• Proteins migrate through pH gradient until their overall charge is neutral

• IEF strip soaked in buffer to impart large negative charge to all proteins (for next step)

Separation Techniques2D-GE - Polyacrylamide Gel Electrophoresis

• Separation of proteins on basis of size

• Small proteins migrate through gel matrix quickest

• Resulting gel has proteins separated– Horizontally by IEP– Vertically by size

Separation Techniques2D-GE - Staining

• Proteins visualised by staining with dyes or metals

• Different dyes have different properties– Silver stain– Coomassie– Fluorescent

Separation Techniques2D-GE - Staining

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1ng 10ng 100ng 1000ng

Separation Techniques2D Gel Electrophoresis

• Limitations– Resolution– Representation– Sensitivity– Reproducibility

• Advantages– Established technology

• Still improving

– Quick– Cheap (relatively)

Separation TechniquesDIGE

• DIfference Gel Electrophoresis

• Variation of standard 2D-GE– Multiple samples on

one gel• Usually 2 samples &

pooled reference– Differentially labelled– Eliminates running

differences between gels

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Separation Techniques2D-GE Analysis

• Gel to Gel comparison identifies varying protein spots

• Images overlaid and examined for differences

• Relies on:– Image warping– Spot matching– Quantitative spot volumes

Separation Techniques2D-GE Analysis

• Progenesis SameSpots (Nonlinear Dynamics)

• DeCyder (GE Healthcare)• Delta2D (DeCodon GmBH)

Separation TechniquesLiquid Chromatography

• Proteins washed through capillary column (or columns)

• Separates based on specific properties– Charge– Size– Hydrophobicity

• Depends on column matrix/eluent

Separation TechniquesLiquid Chromatography

• Usually 2 (or more) columns used (MDLC)

• Can be coupled to Mass Spec (online)• Or fractions collected for later

analysis (offline)• Example: MudPIT (Multidimensional

Protein Identification Technology)

Separation TechniquesLiquid Chromatography

• Limitations– No Peptide Mass Fingerprint

• Protein ID by MS/MS

– Expensive– Difficult

• Advantages– Resolution– Representation– Sensitivity– Reproducibility

Separation TechniquesiTRAQ

• Protein samples digested and labelled

• Labels have different MW reporters

• Differently labelled peptides elute from column together

• MS/MS allows relative abundance of 2 reporters to be calculated

Sample 1 digest

Sample 2 digest

+ Tag + Tag

Reporter Moiety

Balancer Moiety

N-hydroxy succinimide esterfor reaction with primary amines (e.g. N-terminus of peptides)

Total m/z of tag - 145

114 116

Calculate abundance of released reporter moiety

Separation TechniquesiTRAQ

Mass SpectrometryThe Basics

• Analytical technique that measures Mass:Charge ratio (m/z) of ions

• Mass Spectrometers consist of 3 parts:– An ion source– A mass analyzer– A detector system

• Only certain types of Mass Spec are used in proteomics– MALDI, SELDI or Electrospray ion sources– Time of Flight, Quadrupole or Fourier Transform mass

analyzers

• Can Mass Spec whole proteins, but usually just peptides

Mass SpectrometryIonisation - MALDI

• Matrix Assisted Laser Desorption/Ionisation• Sample is mixed with matrix and allowed to

crystallise on a plate• Laser fired at matrix (~100x) produces ions• Typical matrix:

– 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid)

– α-cyano-4-hydroxycinnamic acid (alpha-cyano or alpha-matrix)

– 2,5-dihydroxybenzoic acid (DHB).

Mass SpectrometryIonisation - Electrospray (ESI)

• Sample in volatile solvent• Introduced to highly charged needle• Forces charged droplets from needle• Solvent evaporation leaves only

charged sample

Mass SpectrometryMass Analysis - Time of Flight

• Ions mobilised by high voltage• Travel through flight tube• Deflected by reflectron (an ‘ion mirror’)

– Increases the path length (often doubles it)– Therefore increases the resolution

• Time taken to reach detector is directly proportional to mass of the analyte

Mass SpectrometryMass Analysis - Time of Flight

Mass SpectrometryMass Analysis - Quadrupole

• 2 different charges applied to 2 pairs of metal rods

• Ions travel down the quadrupole between the rods

• Only ions of a certain m/z will be able to travel between the rods for a given charge ratio– Other ions will collide with the rods

• Spectrum produced by scanning voltages

Mass SpectrometryMass Analysis - Quadrupole

Mass SpectrometryMass Analysis - Fourier Transform

• Fourier transform ion cyclotron resonance

• Determines m/z based on cyclotron frequency of ions in a fixed magnetic field

• Ions do not hit the detector, but are sensed as they pass close to it

• Produces a frequency spectrum– A Fourier Transform procedure produces the

mass spectrum from this

Mass SpectrometryMass Analysis - Fourier Transform

Mass SpectrometryTandem MS

• Multiple mass analysis steps• Separated by fragmentation• Multiple methods of fragmenting

– collision-induced dissociation (CID)– electron capture dissociation (ECD)– electron transfer dissociation (ETD)– chemically assisted fragmentation

(CAF)

Protein IdentificationPeptide Mass Fingerprinting

• Proteases cut at defined sites– e.g. trypsin cuts C-terminal of K or R

• Proteins cut with an enzyme will give a series of peptides of different masses

• Different proteins will give different series of peptides

• This is the peptide mass fingerprint of a protein

Protein IdentificationPeptide Mass Fingerprinting

• Alcohol dehydrogenase (374aa, human) gives 26 peptides greater than 500 Da

– 5795.795, 2861.4138, 2836.509, 2294.2069, 1685.9261, 1649.8493, 1645.8076, 1583.8315, 1557.7804, 1277.6228, 1181.7404, 1001.4833, 955.4731, 944.52, 920.5451, 889.4737, 885.5404, 846.4866, 827.4257, 780.4072, 695.2599, 648.3311, 622.3229, 580.3341, 573.2878, 564.281, 548.2787

• Guanine Nucleotide-Binding Protein, alpha-15 (374aa human) gives 31 peptides greater than 500 Da

– 3856.7945, 2092.0498, 1890.9748, 1864.0254, 1826.9734, 1769.8275, 1717.7924, 1690.8646, 1512.7263, 1360.6491, 1343.5606, 1326.5163, 1301.7212, 1295.6353, 1121.6565, 1083.6408, 1058.5339, 992.5299, 950.4434, 873.4424, 847.4407, 815.4621, 743.4661, 732.3522, 724.3876, 701.3253, 662.362, 660.3675, 595.345, 531.2885, 503.2936

• If you look at the two lists of peptide masses you will not see any matches

Protein IdentificationPeptide Mass Fingerprinting

• Alcohol dehydrogenase 7 (374 aa, human) gives 26 peptides greater than 500 Da

– 5795.795, 2861.4138, 2836.509, 2294.2069, 1685.9261, 1649.8493, 1645.8076, 1583.8315, 1557.7804, 1277.6228, 1181.7404, 1001.4833, 955.4731, 944.52, 920.5451, 889.4737, 885.5404, 846.4866, 827.4257, 780.4072, 695.2599, 648.3311, 622.3229, 580.3341, 573.2878, 564.281, 548.2787

• Alcohol dehydrogenase beta2 (375 aa, human) gives 25 peptides greater than 500 Da

– 4256.1078, 2846.4471, 2211.097, 1945.951, 1758.8003, 1729.9523, 1580.7261, 1555.8366, 1329.6797, 1202.6602, 1067.4826, 954.5982, 943.5094, 915.5298, 894.4753, 885.5404, 847.4268, 798.4144, 785.39, 637.3304, 594.2916, 580.3341, 543.3137, 526.2442, 516.2888

• Two closely related protein and yet only two peptides match

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Protein IdentificationPeptide Mass Fingerprinting

699.45544, 896.32411, 909.51544, 909.75215, 912.58639, 920.50129, 973.56255, 1120.58328,

1127.71575, 1193.71203, 1508.56263, 1524.83725, 1525.14491, 1581.85175, 1718.0056, 1721.99879,

1979.20465, 2161.18785, 2184.04418, 2185.00575, 2201.3252, 2514.47913, 3354.92129, 3358.93766

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Deisotoping and Noise Reduction

Extract Peak List

Database Search

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Protein IdentificationMS/MS

• Peptides fragment in a predictable way

• From an MS/MS spectrum, you can work out the peptide sequence

• A peptide of >7 amino acids should be sufficient to uniquely identify a protein

Protein IdentificationMS/MS

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Parent ion m/z = 1522.64

Daughter ion spectra can be deconvoluted to give sequence. The major PMF search engines can also achieve protein ID by MS/MS (MASCOT, SEAQUEST etc).

Role of Bioinformatics

• Software packages for image analysis are complicated– A large part of my job is training lab

biologists to use them– Now moving into LC/MS analysis too

• Downstream analysis of experiments– Similar in many ways to microarrays– Visualisation of results can aid understanding

• Data standards– MIAPE, PSI, HUPO… more about this later

Summary

• Most proteomics experiments have same skeleton – Purification, Separation, Identification

• Many different technologies– 2DGE, LC, MALDI, SELDI, TOF, FT etc

• Importance of bioinformatics increasing

Any questions?

After the fact questions:[email protected]