Download - Lecture 11 Some q uantitation methods with LC-MS ICAT iTRAQ P roteolytic 18 O labelling SILAC
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Lecture 11Some quantitation methods with LC-MSa.ICATb.iTRAQc.Proteolytic 18O labellingd.SILACe.AQUAf.Label Free quantitation
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Thermo scientific webpage
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ICAT
http://www.imsb.ethz.ch/researchgroup/rudolfa/research
Isotope-Coded Affinity Tags
This part reacts with cysteine
The D-labelled (heavy) reagent witll be 8 mass units heavier than the H-labelled (light) reagent
This part binds specificallyto an avidin affinity column
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ICAT
The heavy and light tags haveidentical chemical propertiesso they will bind to any columnin exactly same manner
Avidin affinity columnbinds specifically to biotinso only ICAT labelled peptides will bind to column
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Separate by ion-exchange and into LC-MS/MS
MS of any peak in the chromatogramTwo peaks differ by 8 mass unitsRatio of light:heavy – tells us relative amountof sample 1 to sample 2
MS/MS of the peak in the MSTo identify the peptide
ICAT
Disadvantages: protein must have cysteinelow 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
N
NCH3
O
NH
O
RAdds tag of mass 145 toterminal NH2 groups and lysines
N
NCH3
CH2+
Rest of molecule +
Reporter ion
MS/MS Fragmentation
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N
NCH3
O
ON
O
O
iTRAQ
Produces an ion of Mw = 117after fragmentation
Mw = 28
N
NCH3
O
ON
O
O
Produces an ion of Mw = 116after fragmentation
Mw = 29
13CO
N
NCH3
O
ON
O
O
Mw = 30
C18O
etc
Produces an ion of Mw = 115after fragmentation
13C x 3 15N x 1
13C x 2 15N x 1
13C x 2
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iTRAQ
http://www.broadinstitute.org/scientific-community/science/platforms/proteomics/itraq
Up to 8 different treatments (different types of iTRAQ reagents) – 4 in this example
http://www.iop.kcl.ac.uk/departments/?locator=1031&context=1235
The peptides will elute at the same timebecause they have identical chemical properties
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The MS corresponding to one particular peak in the chromatogramSelect one peak for further fragementation by MS/MS
iTRAQ
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This part gives the sequence information
The low molecular weight region 114-117 contains reporter ionsRatio tells us something about the relative abundance of this proteinin the 4 samples
iTRAQ
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Proteolytic 18O labellling
RNH
NHNH
NH
O
O
O
O
R
R1
R2
R3
If R2 = lysine, arginine
trypsin
H218O
RNH
NH
O
O
OR1
R2
OH
*
* Carboxy oxygens replacedby heavy oxygen
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Proteolytic 18O labellling
Quantitative Protein Analysis Using Proteolytic [18O]Water LabelingKristy 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
Labelling arginine and lysine to ensure all tryptic peptides are labelled (Trypsin cuts at K or R)
Typically e.g. Lys4 – alkly Dx4 subsitution – +4 unitsArg6 – 13Cx6 subsitution – +6 unitsLys8 - 13Cx6 + 15Nx2 substitution +8 unitsArg10 - 13Cx6 + 15Nx4 substitution +10 units
NH
O
NH
NH2
NH2
OH
O
NH2
NH2OH
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SILAC – stable isotope labelling of amino acids in cell culture
Lyse, extract protein, separatetrypsin digest, MS
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AQUA – absolute quantification with reference peptides
Create an isotopically labelled peptide similar to one found in the sampleand spike into the sample
Make an isotopically labelled peptide – in this case Leucine – 13Cx6 +
15Nx1 – +7 mass units
Select a peptide to monitor
Subject this peptide to MS/MSto look at fragmentation pattern
See next slide
(ignore the phosphoprotein part for this lecture)
Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS.Gerber SA, Rush J, Stemman O, Kirschner MW, Gygi SP.Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):6940-5
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AQUA – absolute quantification with reference peptides
1st Quad – allows only one precursor ion through
2nd Quad – trap ions for Collision Induced Decomposition
3rd Quad – allows only one product ion through to be detected
SRM – selective ion monitoring
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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 monitorm/z 374.8 → 564.2 to monitor the natural peptide in samplethen m/z 378.3 → 564.2 to monitor the spiked heavy peptideCompare the peak areas to quantitate peptide in sample
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Label-free Quantitation
LC-MS/MS chromatogram
Assume that a protein X is tryptically digested and the peptides elute aspeaks A (12.28 min), B (14.94 min), C (19.72 min) and D (22.69)
A
B CD
Two examples of label-free quantitationXIC – extracted ion chromatogramSC – spectral counting
Avoid isotopes but instrumentation needs to be very reproducible
E
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Label-free Quantitation - XIC
Shows that peak A represents several peptides co-elutingThe peak area of peak A in the chromatogram is an addition of the peak areas in the MS
e.g. the MS corresponding to peak A
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 Ain 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)
Only one peak bec none of the other peptides have a molecular mass of 660.96Find the peak area
Repeat the same process for all the other peaks B, C and DSum all the peak areas in the XICsConcentration 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 highestso selected for MS/MS
Use e.g. protein Prospector to determine that this peptide sequence is derived from protein X
[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
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Peak at 2116.8 is 3rd highest so selected for MS/MS
MS of peak B
Repeat for all the MS of each peaks
Use e.g. protein Prospector to determine that this peptide sequence is derived from protein X
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)
Label-free Quantitation – SC