introduction to proteomics phil charles ccmp. overview of talk overview of proteomics as a concept...
TRANSCRIPT
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Introduction to Proteomics
Phil CharlesCCMP
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Overview of Talk
• Overview of proteomics as a concept• Techniques discussion• 2D Gels and experimental design
paradigms• Proteomics mass spectrometry• Identification• Quantitation
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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• …
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A Different View on Life
• Different levels of biological complexity
• More layers of regulation and control• Increased heterogeneity of samples
Genome Transcriptome Proteome … Phenotype
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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.
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Proteomics
• Classical biochemistry• Two-dimensional gels (2DGE)• Mass spectrometry• Computational analysis
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Methods in Proteomics
• Separation– Gels– Immunochemistry– Chromatography
• Identification– Immunochemistry–Mass spectrometry
• Quantitation– All of the above
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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
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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.
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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
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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
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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.
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Normalised Abundance Normalised Abundance Normalised Abundance
Statistical Analysis
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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. ’
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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’
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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
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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
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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
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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
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A brief overview of Mass Spectrometric quantitation
Please feel free to stop me and ask questions!
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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
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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)
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Intensity
Retention Time
m/z
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Intensity
Retention Time
m/z
Intensity
m/z
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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
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Intensity
Retention Time
m/z
Intensity
m/z
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Intensity
Retention Time
m/z
m/z
Intensity
IITHPNFNGNTLDNDIMLIK
Intensity
m/z
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Quantitation Labelling Strategies
• MS-based strategies– In-vivo labelling (compare peak pairs) • SILAC, 15N, 18O, 2H
• MS/MS-based strategies– Isobaric Tags• iTRAQ, TMT
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Intensity
Retention Time
m/z
Intensity
m/z
m/z
Intensity
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Intensity
Retention Time
m/z
Intensity
m/z
m/z
Intensity
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Isobaric Tag Labels e.g. iTRAQ, TMT
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Intensity
Retention Time
m/z
m/z
Intensity
IITHPNFNGNTLDNDIMLIK
Intensity
m/z
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Intensity
Retention Time
m/z
Intensity
m/z
m/z
Intensity
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Intensity
Retention Time
m/z
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Intensity
Retention Time
m/z
MS quantitation - peak pair comparison
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Intensity
Retention Time
m/z
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Intensity
Retention Time
m/z
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Intensity
Retention Time
m/z
ID
ID
ID
ID
ID
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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
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Quantitation Software
• MaxQuant• Progenesis LC-MS• ABI Peaks• Thermo ProteomeDiscoverer• + bespoke and specific tools
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The Oxford Central Proteomics Facility
• CCMP/CPF – Kessler Lab – WTCHG• CPF - Ben Thomas – Dunn School
• Computational Biology Research group - WIMM
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Thank you for your attention
Please feel free to ask questions