1. High Resolution Isolelectric Focussing - HiRIEF Group 4

4. What we did in the lab

5. Cell lysates Denaturing proteins Reduction Alkylation Tryptic digestion SCX cleaning Sample preparation

6. Fractionation step Resuspend peptides in rehydration solution Swell gel-strips with peptide solution overnight Run IPG-strips in the machine overnight Extraction of 72 fractions from the IPG-strip

7. Run LC- MS No frac. 1.Peptide from SCX cleaning directly HiRIEF 2. Extracted peptide after fractionation

8. Fractions pooled: 18pH 4.75 21 24 27 30 33 36 39 42pH 7.074

9. First 3 fractions

10. Middle 3

11. Last 3

12. Identified peptides – HiRief vs. no frac.

13. Identified proteins – HiRief vs. no frac.

14. Summary HiRiefNo Frac. Coverage7%13.7% Unique peptides60176520 Shared peptides199 (3%)380 (5%) Total peptides62166900

15. Identified Phosphopeptides

16. Misscleavages HiRIEF No frac.

17. TOP 10 hits No frac.: AreaPSM [ACTB_HUMAN][AHNK_HUMAN] [ACTBL_HUMAN][FLNA_HUMAN] [ACTH_HUMAN][ACTB_HUMAN] [POTEE_HUMAN][MYH9_HUMAN][K2C8_HUMAN] [TBA1B_HUMAN][PLEC_HUMAN] [TBA1C_HUMAN][ENOA_HUMAN] [G3P_HUMAN][HS90B_HUMAN] [H2B1K_HUMAN][FLNB_HUMAN] [H2B1J_HUMAN][K1C18_HUMAN] HiRief.: AreaPSM [ACTB_HUMAN][G3P_HUMAN] [ACTA_HUMAN][ACTB_HUMAN] [H32_HUMAN][FLNA_HUMAN] [ANXA2_HUMAN][PGK1_HUMAN] [FLNA_HUMAN][ENOA_HUMAN] [G3P_HUMAN][ANXA2_HUMAN] [PRDX1_HUMAN][K1C17_HUMAN] [HSP7C_HUMAN][TKT_HUMAN] [K1C17_HUMAN][AHNK_HUMAN] [NDKB_HUMAN][HSP7C_HUMAN]

18. GO term enrichment – DAVID: HiRief Top 100

19. Pathway enrichment – REACTOME: HiRief Top 100

20. GO term enrichment – DAVID: No fractionation Top 100

21. GO term enrichment – DAVID: No fractination Top 100

22. Shared proteins of GO enrichment – Top 100

23. Quantification No internal standard/labels Synthetic peptides, iTRAQ, TMT, SILAC Possible quantification: Area PSM

24. Protein isolation with HiRIEF Pros Reproducible Decrease complexity More sensitive Cons Missing peptides with PI out of the pH range Extra lab step Higher input necessary

25. Did you find any proteins that you are interested in for your own research? Mff Mfn2 MRP3 MRP1 IL18