1. Oct 2011 SDMBT1 Lecture 11 Some quantitation methods with LC-MS a.ICAT b.iTRAQ c.Proteolytic 18 O labelling d.SILAC e.AQUA f.Label Free quantitation

2. Oct 2011 SDMBT2 Thermo scientific webpage

3. Oct 2011 SDMBT3 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 specifically to an avidin affinity column

4. Oct 2011 SDMBT4 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

5. Oct 2011 SDMBT5 Separate by ion-exchange and into LC-MS/MS 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 MS/MS of the peak in the MS To identify the peptide ICAT Disadvantages: protein must have cysteine low sequence coverage – protein is identified often based on only one peptide, often not able to identify PTM

6. Oct 2011 SDMBT6 iTRAQ Isobaric Tag for relative and absolute quantification Reacts with NH 2 groups Adds tag of mass 145 to terminal NH2 groups and lysines Rest of molecule + Reporter ion MS/MS Fragmentation

7. Oct 2011 SDMBT7 iTRAQ Produces an ion of Mw = 117 after fragmentation Mw = 28 Produces an ion of Mw = 116 after fragmentation Mw = 29 13 CO Mw = 30 C 18 O etc Produces an ion of Mw = 115 after fragmentation 13 C x 3 15 N x 1 13 C x 2 15 N x 1 13 C x 2

8. Oct 2011 SDMBT8 iTRAQ http://www.broadinstitute.org/scien tific- community/science/platforms/prot eomics/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 time because they have identical chemical properties

9. Oct 2011 SDMBT9 The MS corresponding to one particular peak in the chromatogram Select one peak for further fragementation by MS/MS iTRAQ

10. Oct 2011 SDMBT10 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 in the 4 samples iTRAQ

11. Oct 2011 SDMBT11 Proteolytic 18 O labellling If R 2 = lysine, arginine trypsin H 2 18 O * *Carboxy oxygens replaced by heavy oxygen

12. Oct 2011 SDMBT12 Proteolytic 18 O labellling Quantitative Protein Analysis Using Proteolytic [18O]Water Labeling Kristy J. Reynolds, Catherine Fenselau, Current Protocols in Protein Science, 2004

13. Oct 2011 SDMBT13 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 units Arg6 – 13 Cx6 subsitution – +6 units Lys8 - 13 Cx6 + 15 Nx2 substitution+8 units Arg10 - 13 Cx6 + 15 Nx4 substitution+10 units

14. Oct 2011 SDMBT14 SILAC – stable isotope labelling of amino acids in cell culture Lyse, extract protein, separate trypsin digest, MS

15. Oct 2011 SDMBT15 AQUA – absolute quantification with reference peptides Create an isotopically labelled peptide similar to one found in the sample and spike into the sample Make an isotopically labelled peptide – in this case Leucine – 13 Cx6 + 15 Nx1 – +7 mass units Select a peptide to monitor Subject this peptide to MS/MS to 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

16. Oct 2011 SDMBT16 AQUA – absolute quantification with reference peptides 1 st Quad – allows only one precursor ion through 2 nd Quad – trap ions for Collision Induced Decomposition 3 rd Quad – allows only one product ion through to be detected SRM – selective ion monitoring

17. Oct 2011 SDMBT17 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 (y 4 ) - set the LC-MS/MS into the SRM mode so that only m/z 378.3 ions are allowed through 1 st Q and 571.2 ions are allowed through the 3 rd Q

18. Oct 2011 SDMBT18 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

19. Oct 2011 SDMBT19 Label-free Quantitation LC-MS/MS chromatogram 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) A B C D Two examples of label-free quantitation XIC – extracted ion chromatogram SC – spectral counting Avoid isotopes but instrumentation needs to be very reproducible E

20. Oct 2011 SDMBT20 Label-free Quantitation - XIC 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. 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 A in the chromatogram Work out the contribution of the 660.96 to the peak area of peak A by

21. Oct 2011 SDMBT21 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 660.96 Find the peak area 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)

22. Oct 2011 SDMBT 22 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 2 nd highest so 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

23. Oct 2011 SDMBT 23 Peak at 2116.8 is 3 rd 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