Transcript How sure are you about the ID?

Introduction to Proteomics
Phil Charles
CCMP
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
Genome  Transcriptome  Proteome  Phenotype
• Different levels of biological complexity
• More layers of regulation and control
• Increased heterogeneity of samples
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
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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
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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 Toussaint
Upregulation of annexin A2 in H2O2-induced
premature senescence as evidenced by 2DDIGE proteome analysis
Experimental 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
Statistical Analysis
Normalised Abundance
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, ‘topdown proteomics’ –
identify and quantify
whole proteins.
• Most modern proteomic
MS is ‘bottom-up’
Shotgun/’bottom-up’ proteomics
LNDLEEALQQAKEDLAR
NKLNDLEEALQQAK
NVQDAIADAEQR
SKEEAEALYHSK
SLVGLGGTK
TAAENDFVTLK
TAAENDFVTLKK
TSQNSELNNMQDLVEDYK
TSQNSELNNMQDLVEDYKK
VDLLNQEIEFLK
YEELQVTVGR
YLDGLTAER
ADLEMQIESLTEELAYLK
ADLEMQIESLTEELAYLKK
AETECQNTEYQQLLDIK
LNDL
EEAL
QQAC
EDLA
R
N
KLND
LEEAL
QQAK
Proteins
Separation Digestion Peptides Separation
SDS-PAGE
SCX
Antibody-based
High pH RP LC
approaches
Low pH RP LC
Analysis
MS-MS/
Tandem MS
Peptide IDs
+ Quantitation
IPI:IPI00000073.2
IPI:IPI00217963.3
IPI:IPI00031065.1
IPI:IPI00376379.4
IPI:IPI00397801.4
IPI:IPI00009950.1
IPI:IPI00395488.2
IPI:IPI00295414.7
IPI:IPI00554711.3
IPI:IPI00009867.3
IPI:IPI00019449.1
IPI:IPI00016915.1
IPI:IPI00060800.5
IPI:IPI00013885.1
IPI:IPI00221224.6
Observed Proteins
+ Quantitation
Mass Spectrum
m/z
Tandem Mass Spectrum
MS/MS spectrum
Mass
Analyser
+
Detector
Intensity
Sample
Mass
Analyser
+
Detector
Intensity
Tandem Mass Spectrometry
m/z
Identification by MS/MS
m/z
?
Intensity
• 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
Intensity
Mass
Analyser +
Detector
m/z
IITHPNFNGNTLDNDIMLIK
Identification by MS/MS
• There are multiple commonly used MS/MS
fragment spectra search engines, including:
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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!
Mass Spectrum
m/z
Tandem Mass Spectrum
MS/MS spectrum
Mass
Analyser
+
Detector
Intensity
Sample
Mass
Analyser
+
Detector
Intensity
Tandem Mass Spectrometry
m/z
Select Peptide
Ions
Fragmentation
Low pH
Reverse
Phase LC
‘Survey Scan’/
‘MS1’/
‘MS Scan’
CID
Also ETD,
PQD,HCD
‘Fragment Ions
Scan’/
‘MS2’/
‘MS/MS Scan’
Data-Dependent
Acquisition (DDA)
time
Intensity
Retention Time
m/z
Intensity
Intensity
Retention Time
m/z
m/z
Peptide Isotopomer Distribution
This is all 1 peptide
Intensity
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
m/z
1/charge
(z)
Intensity
Intensity
Retention Time
m/z
m/z
Intensity
Intensity
Intensity
m/z
Retention Time
m/z
IITHPNFNGNTLDNDIMLIK
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
Intensity
Intensity
m/z
Retention Time
m/z
m/z
Intensity
Intensity
Intensity
m/z
Retention Time
m/z
m/z
Isobaric Tag Labels e.g. iTRAQ, TMT
Intensity
Intensity
Intensity
m/z
Retention Time
m/z
IITHPNFNGNTLDNDIMLIK
m/z
Intensity
Intensity
Intensity
m/z
Retention Time
m/z
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
Intensity
Retention Time
m/z
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
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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