Workshop in Quantitative 'Omics Technologies
Edinburgh 5th November 2009
Quantitative Proteomics
Kathryn Lilley
Cambridge Centre for Proteomics
Department of Biochemistry
University of Cambridge
k.s.lilley@bioc.cam.ac.uk
www.bio.cam.ac.uk/proteomics/
Complexity
What does the protein
do?
Where is the protein
located?
Which other proteins
are its binding
partners?
Is the protein posttranslationally
modified?
Types of Proteomics
Experiments
Cataloguing:
Binding
Comparative
Partners:
/state:
Quantitative:
Modification
What
What
are
are
changes
the members
inmodifications
abundance
of protein
proteins
complexes?
between
proteins
are
present
in aofand
given
sample?
Post
translational
how
do
these
two or more
samples?
change
between
samples?
Quantitative Proteomics
Relative
Absolute
Relative Quantitation
The Tools Required
Mass Spectrometers
• Mass spectrometers all contain:
– Source where ionisation takes place
– Mass Analyser – ions in the gaseous phase
are separated based on their mass/charge
ratio (m/z)
– Detectors - measures m/z
Source I: Electrospray Ionisation (ESI)
Molecules are sprayed through a fine capillary
The high electrostatic potential causes the sample to be sprayed as
charged micro-droplets
The drying gas (N2) causes evaporation of solvent and ion formation
Source II: Matrix Assisted Laser
Desorption Ionisation (MALDI)
Ion selection and analysis
Quadrupole
3D ion trap
Orbitrap
Time of flight detector
MALDI Tof MS
Reflectron
Detector
Matrix
Suppression
Lens
Sample target
Gas
Cell
Ion Beam
N2 Laser
Reflectron
Assembly
Linear
Detector
The chemical matrix absorbs energy from the laser pulse which is transferred to the protein
The sample ions are then accelerated towards the detector
Principally produces M+H+ ions (sometimes M+2H+ )
Hybrid Instruments
Quadrupole Tof MS (QTof)
MS – measure m/z
MS/MS – select m/z
fragment
measure m/z of fragments
Stage 1 – selection in quadrupole
Stage 2 - fragmentation in hexapole
Stage 3 – analysis in TOF
LTQ-Orbitrap MS
Overview of Protein
Identification by MS
• Protein/peptide separation techniques
• Protein identification
• Protein sequencing
Separations at the protein level
GelCMS
Proteolytic digestion
MS
2D PAGE
Proteolytic digestion
MS
Separations at the peptide level
Protein(s)
+
=
Peptides
Trypsin
Single dimension
Reverse Phase HPLC
Proteolytic digestion
Multi dimensional (MudPIT)
Strong cation exchange
Reverse Phase
Proteolytic digestion
HPLC coupled to a mass spectrometer or MALDI spotter
• Protein/peptide separation techniques
• Protein identification
• Protein sequencing
Peptide Mass Fingerprinting
K
R
R
Trypsin
Peptides
K
Matrix assisted desorption time
of flight mass spectrometry
Identification !!!!
Database search of virtual trypsin
digested translated genome
1457.35
1765.33
1975.72
2055/78
2589.31
Mass list
100 kDa toxin receptor from M. sexta
• Protein/peptide separation techniques
• Protein identification
• Protein sequencing
Tandem Mass Spectrometry
a.k.a.
de novo sequencing
LC-MS/MS
• Using e.g. Triple quadrupole, Ion-trap or Q-TOF MS
ara 11b
Plate Flatness
jh150301b 5 (1.140) Cm (1:5)
100
15-MAR-2001
12:06:58
TOF LD+
3.16e4
1676.97
1498.86
2173.00
2172.01
1762.80
1499.86
1994.96
1993.96
1475.74
2174.04
%
1995.97
1763.78
1500.86
1765.76
1179.60
1277.71
1766.75
1303.67
2213.14
1839.98
1501.86
1119.50
888.52
2212.14
1996.97
1638.87
834.42
2706.15
2511.13
2567.20
2510.17
2924.38
0
m/z
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
(M+2H)2+
749.912
100
ESI-MS/MS
888.514
1102.591
298.170
197.130
%
413.198 512.265
700.373
169.129
750.904
889.509
70.068
987.582
801.477
V V S V
D
72.085
890.517
1302.746
D V
T
891.526
P
1305.686
0
m/z
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
Results:
Some peptides detected by MS:
Relative Quantification
2D PAGE
Isotope Labelling
Label Free
PROTEIN
PEPTIDE
PEPTIDE
In vivo
Single sample per gel
Coomassie
Sypro Ruby etc…
Silver
Multi samples per gel
DIGE
SILAC
14/15N 12/13C
In vitro
iTRAQ
TMT
H216/18 O
methylation
Peak intensity
Spectral counting
Difference Gel Electrophoresis
(DIGE)
• First described by Jon Minden (Carnegie
Mellon University. Pittsburg, USA
– Ünlü M. et al (1997). Electrophoresis,18, 2071-2077
Sample 1
label with cy2
in dark 30mins @ 4OC
Sample 2
Sample 3
label with cy3
in dark 30mins @ 4OC
label with cy5
in dark 30mins @ 4OC
quench un-reacted dye
by adding 1mM lysine
in dark 10mins @ 4OC
•Ünlü M. et al (1997). Electrophoresis,18, 2071-2077
2D gel electrophoresis
Cy5
Cy3
no difference
●
presence / absence
●●
up / down-regulation
●
Cy3 +Cy5
Standardised log abundance
0.4
0.3
0.2
r
P ai
0.1
Increase
1
Pair 2
P ai
0
No change
r3
Decrease
-0.1
Schizophrenia
Control
-0.2
Allows many many gels to be linked in an experiment
involving multiple samples
2D Gel Approach Summary
• Strengths
–
–
–
–
–
Quick
Accessible to all
Relatively inexpensive hardware (software £££££ however)
Visualisation of isoforms with differing pI’s or MW’s
Sample storage
• Weaknesses
Only proportion of proteome visualised
– Following poorly represented:
Membrane proteins
Proteins with extremes of pI
Proteins with extremes of MW
– Resolution
Co-migration
– Low abundance species (1000-5000 copies per cell)
90% protein product of 10% mammalian genome
Quantification using differential
stable isotope labelling
In vivo
Elemental
Amino acid
(SILAC)
In vitro
MS
MS/MS
Stable Isotope Labelling – in
vivo
Sample 1
Sample 2
incorporates
natural isotope
incorporates
heavier isotope
Digest with protease
MS/MS to identify
Mixture of light/heavy peptides
LC separation – usually multi dimensional
light
heavy
Quantitation in MS
Stable Isotope Labelling – in vivo
Elemental
?
– Samples grown in medium
where there is replacement of an
element with a stable isotope
– Typically 15N instead of 14N, or
13 C instead of 12C
–
13
C not often used as more
carbon in proteins than nitrogen
and therefore big mass shifts
– Do not known mass difference
between light and heavy pairs
unless sequence is deduced
(retention times)
Types of samples suitable?
Bacterial / Cell culture
E coli grown on 15 N sole nitrogen source
and then fed to C.elegans
Light mutant
Heavy WT
Krijgsveld et al (2003) Nat. Biotech.21:927
Light WT
Heavy mutant
Stable Isotope Labelling – in vivo
Amino acid - SILAC
(Stable Isotope Labeling with
Amino acids in Cell culture)
– Samples grown in medium
where there is replacement of an
amino acid with heavier stable
isotopic form of the amino acid
– Typically 13 C instead of 12C –
labelled lysine, arginine or
leucine
– Know the mass difference
between light and heavy pairs
– Need to check for extent of
incorporation
Types of samples suitable?
Bacterial / Cell culture
– Conversion to proline a problem
Strengths
Weaknesses
SILAC
Samples to be compared are
combined early in early in
experiment
Information-rich
Can be targeted to identify
peptides which show
change in abundance
cell culture
amino acid metabolism
Metabolic labelling
Limit to applicability
complex data analysis
Complex mixtures of peptides
No PTM information without effort!
Stable Isotope Labelling – in vitro
Analysis at MS stage
•
Many variants including
•
Isotopes introduced during
proteolysis 18O – labelled
water, C-termini
•
Guanidation of lysine using
isotopes of O-methyl isourea
– lysine residues
•
Dimethyl labelling – lysine
residues
•
Mostly the above lead to
small mass differences
16O
18O
Stable Isotope Labelling – in vitro
Analysis at MS/MS stage
iTRAQ reagents (Amine Modifying
Labeling Reagents for Multiplexed
Relative and Absolute Protein
Quantitation)
• 4 x isobaric tags - all 145 Da
• React with primary amines
• Label at peptide level
• Fragment during MSMS to produce
characteristic reporter ion for each tag
Ross et al (2004) Mol. Cell. Prot. 3:1154
Stable Isotope Labelling in vitro iTRAQ
–
Isotopic Variation
-
MS
MSMS
Multiplexing iTRAQ Reagents.
• 8plex Reagent
Strengths
Weaknesses
MS analysis
Information-rich
Suitable for any sample
Small mass differences
for some methods
MSMS – iTRAQ
Require MS and MSMS
in same experiment
Some side reactions (Tyr)
Complex mixtures of peptides
No PTM information without effort!
Quantitation using a label free
approach
Peak measurements
Spectral counting
Compare peak intensities of the
same ion in consecutive LCMS runs
Number of non-redundant spectra
matching the same proteins
Need to match retention times with
m/z values
The number of redundant peptides
observed correlates with abundanc
Strengths
Information-rich
Weaknesses
Peak intensity
Need high performance instruments
Suitable for any sample
Cheap (may need to run more samples)
No pooling of samples,
therefore potential for high degree
of variance
Ion suppression
Spectral counting
Not all proteins digest to same
degree with proteases
Insensitive to small fold changes
in expression
Complex mixtures of peptides
No PTM information without effort!
Absolute Quantitation
Assay proteins of interest
MS based absolute quantitation works by measuring
peptide ‘surrogate’ simultaneously against quantified
internal standard.
Surrogate = peptides
The ions that are used for measurement are
generally MS/MS fragment ions which are
discriminatory for the peptide of choice
Selected Reaction Monitoring
Q
Precursor ion selected
Natural peptide
CID
Q
Diagnostic fragment ions selected
Collision
= transitions
Induced
Fragmentation
m/z
Synthetic version of peptide
containing stable isotope
How to create good peptide
internal standard?
• AQUA
– Gerber et al (2003) PNAS 100(12):6940-5
• QconCAT
– Beynon et al (2005) Nat. Methods 2(8):587-9.
• Labelled proteins ‘mass Western’
– Lehmann et al (2008) The Plant Journal 55:1039–
1046
AQUA
Stable isotope tagged
synthetic peptide
protein of interest
Assumption: Stoichiometric release of peptide surrogate.
Internal standard not generated by tryptic cleavage
Tryptic digestion
QconCAT
Stable isotope labelled synthetic protein
Constructed from concatenated peptides(Qprotein)
Protein of interest
Assumption: Stoichiometric release of peptide surrogate.
Internal standard not generated by identical tryptic cleavage
Tryptic digestion
Recombinant labelled protein
Mass Western
Assumption:
Stable isotope labelled recombinant protein
Protein of interest
Identical tryptic cleavage for internal standard and
surrogate.
Complete set of internal standards
Tryptic digestion
Quantitative Proteomics
• Relative quantitation
– 2D gel based
– Differential stable isotope labelling
– MS based label free
• Absolute
– Spiked internal standard of known amount
• Stable isotope labelled protein
• Stable isotope labelled peptides
• Thank you for listening!!!!