4. Methanol/Chloroform precipitation In-solution digestion Off-line HPLC-RP Basic pH Methanol/Chloroform precipitation In-solution digestion Off-line HPLC-RP Basic pH 5600-Ttof (CID) Cut bands Automatic In-gel digestion Digestor (Bruker) 30 fractions / pooled into 10 HPLC-RP Acid pH 15 bands 1D-SDS PAGE RIPA CHAPS/UREA 4% SDS

7. Good overlapping between replicate experiments for the Gel or off-line basicRP prefractionation methods.

9. For all the cell-lysing conditions tested, the in-solution digestion-basicRP-LC/MS workflow was by far the most compatible (between 20-30% more proteins identified).

12. HPLC-RP 1D SDS-PAGE 2 Replicates  Different cell-lysis conditions gave similar proteome coverage in the Gel-LC-MS workflow.  CHAPS lysis enabled greater protein identification in the basic-RP-LC/MS workflow.  The excess of detergents that would alter protein precipitation and digestion.  Different cell-lysis conditions gave similar proteome coverage in the Gel-LC-MS workflow.  CHAPS lysis enabled greater protein identification in the basic-RP-LC/MS workflow.  The excess of detergents that would alter protein precipitation and digestion.

13. 2 Replicates 1D SDS-PAGEHPLC-RP  Overall, more than 4 peptides were identified per protein.  In the off-line HPLC separation, CHAPS lysis provided greater peptide recovery and protein coverage.  Overall, more than 4 peptides were identified per protein.  In the off-line HPLC separation, CHAPS lysis provided greater peptide recovery and protein coverage.

14. More exclusive proteins identified by CHAPS lysis combined with in-solution digestion/off-line HPLC separation. 1D SDS-PAGEHPLC-RP 2 Replicates

17.  CHAPS lysis was better in recovering most sub-cellular compartments even for membrane proteins.  However, our ability to identify membrane proteins is low and we should considerate using plasma membrane enrichment methods (subcellular fractionation, cell-surface biotinylation…)  CHAPS lysis was better in recovering most sub-cellular compartments even for membrane proteins.  However, our ability to identify membrane proteins is low and we should considerate using plasma membrane enrichment methods (subcellular fractionation, cell-surface biotinylation…)

19. ACETYLATION PHOSPHORYLATION TOTAL: 1317 P-Peptides / 865 P-Proteins Chr 16: 66 P-Peptides / 42 P-Proteins TOTAL: 1317 P-Peptides / 865 P-Proteins Chr 16: 66 P-Peptides / 42 P-Proteins Jurkat CHAPS HPLC-RP Replicate 2 Average of 3% of peptides found acetylated Chr 16: 56 peptides N-term acetylated Chr 16: 56 peptides N-term acetylated

21. FDR 1% Protein level Replicate 2 Jurkat TOTALChr 16 IMPORTANTE: Exportar datos siempre de la misma forma

22. Replicate 2 Jurkat FDR 1% Protein level

24. Jurkat CHAPS HPLC-RP 10 fractions Replicate 1 TToF 5600 CNB Q-Exactive UPV Replicate 2 TToF 5600 CNB CHAPS/UREA 10 Fractions

25. FDR 1% Protein level Both mass spectrometers are comparable TOTALChr 16 Jurkat CHAPS HPLC-RP

26. FDR 1% Protein level TOTALChr 16 Reproducibility

27. FDR 1% Protein level TOTALChr 16 Jurkat CHAPS HPLC-RP

29. Adán Alpízar Silvia Juárez Sergio Ciordia Marisol Fernández Adán Alpízar Silvia Juárez Sergio Ciordia Marisol Fernández Rosana Navajas Severine Gharbi Miguel Marcilla Alberto Paradela Carmen González Gonzalo Martínez Rosana Navajas Severine Gharbi Miguel Marcilla Alberto Paradela Carmen González Gonzalo Martínez Alberto Medina Salvador Martínez Antonio Ramos Miguel Ángel López Alberto Medina Salvador Martínez Antonio Ramos Miguel Ángel López Virginia Pavón Lola Segura Virginia Pavón Lola Segura Mª Carmen Mena Fernando Roncal Manuel Lombardía Mª Carmen Mena Fernando Roncal Manuel Lombardía