Lecture 11
Some quantitation methods with LC-MS
a.
b.
c.
d.
e.
f.
ICAT
iTRAQ
Proteolytic 18O labelling
SILAC
AQUA
Label Free quantitation
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1
Thermo scientific webpage
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2
ICAT
Isotope-Coded Affinity Tags
The D-labelled (heavy) reagent witll be
8 mass units heavier than the H-labelled
(light) reagent
This part binds specifically
to an avidin affinity column
This part reacts with cysteine
http://www.imsb.ethz.ch/researchgroup/rudolfa/research
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ICAT
The heavy and light tags have
identical chemical properties
so they will bind to any column
in exactly same manner
Avidin affinity column
binds specifically to biotin
so only ICAT labelled peptides
will bind to column
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ICAT
MS of any peak in the chromatogram
Two peaks differ by 8 mass units
Ratio of light:heavy – tells us relative amount
of sample 1 to sample 2
Separate by ion-exchange and
into LC-MS/MS
MS/MS of the peak in the MS
To identify the peptide
Disadvantages:
protein must have cysteine
low sequence coverage – protein is identified often based on only
one peptide, often not able to identify PTM
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iTRAQ
Isobaric Tag for relative and absolute quantification
Reacts with NH2 groups
R
NH
Adds tag of mass 145 to
terminal NH2 groups and lysines
N
N
O
O
H 3C
+
MS/MS Fragmentation
N
Rest of molecule +
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CH2
N
H 3C
Reporter ion
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iTRAQ
13C
x 3 15N x 1
Mw = 28
O
O
N
N
13C
N
x2
O
O
H 3C
O
O
N
N
Produces an ion of Mw = 117
after fragmentation
13C
C18O
N
O
H 3C
13CO
x 2 15N x 1
O
O
Mw = 30
O
N
N
Produces an ion of Mw = 115
after fragmentation
N
O
H 3C
O
Mw = 29
Produces an ion of Mw = 116
after fragmentation
etc
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iTRAQ
http://www.broadinstitute.org/scien
tificcommunity/science/platforms/prot
eomics/itraq
Up to 8 different treatments (different types of iTRAQ reagents) – 4 in this example
The peptides will elute at
the same time
because they have
identical chemical
properties
http://www.iop.kcl.ac.uk/departments/?locator=1031&context=1235
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iTRAQ
The MS corresponding to one particular peak in the chromatogram
Select one peak for further fragementation by MS/MS
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iTRAQ
This part gives the sequence information
The low molecular weight region 114-117 contains reporter ions
Ratio tells us something about the relative abundance of this protein
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in the 4 samples
10
Proteolytic 18O labellling
R
O
1
R
O
3
trypsin
R
NH
NH
R
NH
NH
O
R
2
H218O
O
If R2 = lysine, arginine
R
O
1
*O
NH
R
*
OH
NH
O
R
Carboxy oxygens replaced
by heavy oxygen
2
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Proteolytic 18O labellling
Quantitative Protein Analysis Using Proteolytic [18O]Water Labeling
Kristy J. Reynolds, Catherine Fenselau, Current Protocols in Protein Science, 2004
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SILAC – stable isotope labelling of amino
acids in cell culture
Grow cells in media containing isotopically labelled amino acids
Typically e.g.
Lys4 – alkly Dx4 subsitution –
Arg6 – 13Cx6 subsitution –
Lys8 - 13Cx6 + 15Nx2 substitution
Arg10 - 13Cx6 + 15Nx4 substitution
+4 units
+6 units
+8 units
+10 units
O
H 2N
OH
NH2
NH2
HN
O
NH
OH
NH2
Labelling arginine and lysine to ensure all tryptic peptides are
labelled (Trypsin cuts at K or R)
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SILAC – stable isotope labelling of amino
acids in cell culture
Lyse, extract protein, separate
trypsin digest, MS
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AQUA – absolute quantification with
reference peptides
Create an isotopically labelled peptide similar to one found in the sample
and spike into the sample
(ignore the phosphoprotein
part for this lecture)
Select a peptide to monitor
Make an isotopically
labelled peptide – in this
case Leucine – 13Cx6 +
15Nx1 – +7 mass units
Subject this peptide to MS/MS
to look at fragmentation pattern
See next slide
Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS.
Gerber SA, Rush J, Stemman O, Kirschner MW, Gygi SP.
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Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):6940-5
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AQUA – absolute quantification with
reference peptides
SRM – selective ion monitoring
2nd Quad – trap ions for
Collision Induced Decomposition
1st Quad –
allows only one
precursor ion
through
3rd Quad – allows
only one product
ion through to be
detected
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AQUA – absolute quantification with
reference peptides
e.g. ALELFR is chosen as the peptide to
monitor
- Synthesize ALEL*FR (+7 mass units)
-do an MS/MS of ALEL*FR
-Choose one of the high intensity
fragment ions to monitor (y4)
- set the LC-MS/MS into the SRM mode
so that only m/z 378.3 ions are allowed
through 1st Q and 571.2 ions are
allowed through the 3rd Q
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AQUA – absolute quantification with
reference peptides
Spike sample with the
synthesised heavy peptide
The heavy peptide will have identical
retention time because chemical
properties are identical
Set the LC-MS/MS to the SRM mode to monitor
m/z 374.8 → 564.2 to monitor the natural peptide in sample
then m/z 378.3 → 564.2 to monitor the spiked heavy peptide
Compare the peak areas to quantitate peptide in sample
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Label-free Quantitation
Two examples of label-free quantitation
XIC – extracted ion chromatogram
SC – spectral counting
Avoid isotopes but instrumentation needs to be very reproducible
LC-MS/MS chromatogram
B
C
E
D
A
Assume that a protein X is tryptically digested and the peptides elute as
peaks A (12.28 min), B (14.94 min), C (19.72 min) and D (22.69)
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Label-free Quantitation - XIC
e.g. the MS corresponding to peak A
Shows that peak A represents several peptides co-eluting
The peak area of peak A in the chromatogram is an addition of the peak areas in
the MS
e.g. if 660.96 is identified by MS/MS as a peptide coming from protein X
– so the peptide from protein X contributes a large part to the peak area of peak A
in the chromatogram
Work out the contribution of the 660.96 to the peak area of peak A by
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Label-free Quantitation - XIC
Work out the contribution of the 660.96 to the peak area of peak A by
extracting the ion chromatogram (XIC)
Find the peak area
Only one peak bec none of the other peptides have
a molecular mass of 660.96
Repeat the same process for all the other peaks B, C and D
Sum all the peak areas in the XICs
Concentration of protein X proportional to total area (need an internal standard)
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Label-free Quantitation – SC
Assuming the LC-MS/MS is set to select only the largest 3 peaks in
the MS for collision to produce MS/MS
MS of peak 2
Peak at 660.96 is 2nd highest
so selected for MS/MS
[all data for the this and the next slide are not taken
from a real example so do not try to use Protein
Prospector to work out the sequence
Use e.g. protein Prospector to
determine that this peptide sequence is
derived from protein X
22
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Label-free Quantitation – SC
Repeat for all the MS of each peaks
MS of peak B
Peak at 2116.8 is 3rd highest so
selected for MS/MS
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Use e.g. protein Prospector to
determine that this peptide sequence is
derived from protein X
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But e.g. for peak E in the
chromatogram, MS/MS of the
top 3 peaks in the MS- come
from different proteins
Count all the MS/MS spectra
which came from a peptide
which can be identified as
coming from protein X
(Spectral Count)
Spectral count proportional
to protein concentration
(need internal standard)