Lecture 11 Some q uantitation methods with LC-MS ICAT iTRAQ P roteolytic 18 O labelling SILAC

1. Lecture 11 • Some quantitationmethods with LC-MS • ICAT • iTRAQ • Proteolytic18O labelling • SILAC • AQUA • Label Free quantitation Oct 2011 SDMBT

2. Thermo scientific webpage Oct 2011 SDMBT

3. 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 Oct 2011 SDMBT

4. 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 Oct 2011 SDMBT

5. 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 Oct 2011 SDMBT

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

7. iTRAQ 13C x 3 15N x 1 Mw = 28 C18O 13C x 2 Produces an ion of Mw = 117 after fragmentation 13CO 13C x 2 15N x 1 Mw = 30 Produces an ion of Mw = 115 after fragmentation Mw = 29 etc Produces an ion of Mw = 116 after fragmentation Oct 2011 SDMBT

8. 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 The peptides will elute at the same time because they have identical chemical properties Oct 2011 SDMBT http://www.iop.kcl.ac.uk/departments/?locator=1031&context=1235

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

10. 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 in the 4 samples Oct 2011 SDMBT

11. Proteolytic 18O labellling trypsin H218O If R2 = lysine, arginine * * Carboxy oxygens replaced by heavy oxygen Oct 2011 SDMBT

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

13. 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 – +4 units Arg6 – 13Cx6 subsitution – +6 units Lys8 - 13Cx6 + 15Nx2 substitution +8 units Arg10 - 13Cx6 + 15Nx4 substitution +10 units Labelling arginine and lysine to ensure all tryptic peptides are labelled (Trypsin cuts at K or R) Oct 2011 SDMBT

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

15. 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. Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):6940-5 Oct 2011 SDMBT

16. 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 Oct 2011 SDMBT

17. 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 Oct 2011 SDMBT

18. 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 Oct 2011 SDMBT

19. 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 C E B 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) Oct 2011 SDMBT

20. 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 Oct 2011 SDMBT

21. 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) 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 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 Oct 2011 SDMBT

23. Label-free Quantitation – SC Repeat for all the MS of each peaks But e.g. for peak E in the chromatogram, MS/MS of the top 3 peaks in the MS- come from different proteins MS of peak B Count all the MS/MS spectra which came from a peptide which can be identified as coming from protein X (Spectral Count) Peak at 2116.8 is 3rd highest so selected for MS/MS Spectral count proportional to protein concentration (need internal standard) Use e.g. protein Prospector to determine that this peptide sequence is derived from protein X Oct 2011 SDMBT