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sHPP

sHPP. Chromosome 16 INIBIC. Miraflores de la Sierra, la Cristalera, 28 th and 29 th August 2012. Quantitation using Bradford and Densitometry. Ramos CCD18 MCF-7 Tc2802. 4 μ g. In solution Digestion. 50 μ g sample TC2802 (3x) 6,5 μ l sample + 3,5 μ l AmBi

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sHPP

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  1. sHPP Chromosome 16 INIBIC Miraflores de la Sierra, la Cristalera, 28th and 29thAugust 2012

  2. Quantitationusing Bradford and Densitometry Ramos CCD18 MCF-7 Tc2802 4 μg

  3. In solutionDigestion • 50 μg sample TC2802 (3x) • 6,5 μl sample + 3,5 μl AmBi • Redutionwith DTT 10 mM 1h, 37ºC • Alquilationwith IAA 50 mM 45 min in thedark • DilutioneithAmBi [Urea<1M] • Digestión con Tripsina (Promega): 2μg/muestra O/N (1:25) Sampleclean up StageTips Poros R2 NuTips • 1. ConditioningProcedure • Metanol 2x • 80% ACN 0,1% TFA 2x • 0,1 % TFA 2x • 2. SampleBinding • 3. SampleWashing • 0,1 % FA 2x • 4. SampleRelease • 80% ACN 0,1% FA 2x • 1. ConditioningProcedure • 80% ACN 0,1 % TFA 10x • 0,1 % TFA 3x • 2. SampleBinding • 3. SampleWashing • 0,1 % FA 5x • 4. SampleRelease • 80% ACN 0,1% FA 2x • 1. ConditioningProcedure • 100% ACN 3x • 0,5% TFA 2x • 0,1 % TFA 2x • 2. SampleBinding • 3. SampleWashing • 0,1 % FA 2x • 4. SampleRelease • 80% ACN 0,1% FA 2x 50μg/50μl of 2%ACN 0.1% FA

  4. NanoLC-1D+, TEMPO (ABSciex) Channel 1 Trap Acclaim Pep Map 100 nano viper 100µm*2cm, C18, 5 µm, 100A F=3 µl/min @10min A /B, 2% ACN, 0.1 Formic acid 50/50 Channel 2 Column Acclaim Pep Map 100 75µm *15cm, C18, 3µm, 100A F=300nL/min LC MRM LC EM ER Q-TRAP 5500 (ABSciex) ProteinPilot software Uniprot_treml_human_chr16 Uniprot_sprot_human_chr16 Nextprot_chromosome_16

  5. QTRAP 5500 EM ER

  6. QTRAP 5500 MRM

  7. EM ER Q-TRAP 5500 R2, chondrocytes

  8. EM ER A Q-TRAP 5500 NUTIPS chondrocytes

  9. EM ER Q-TRAP 5500 STAGE TIPS, chondrocytes

  10. EM ER A Q-TRAP 5500 3 clean-ups

  11. RESULTS EM ER FUS

  12. RESULTS EM ER CPSF5

  13. MRM PILOT SELECTION CRITERIA MRM PILOT TRANSITIONS + SRM PEPTIDE ATLAS

  14. PEPTIDE ATLAS

  15. MRM PEPTIDE ATLAS Q-TRAP 5500

  16. MRM RESULTS Q-TRAP 5500, chondrocytes

  17. MRM RESULTS Q-TRAP 5500, chondrocytes

  18. MRM RESULTS Q-TRAP 5500, chondrocytes

  19. MRM RESULTS Q-TRAP 5500, chondrocytes

  20. MRM CONCLUSIONS Q-TRAP 5500, chondrocytes

  21. TIC FUS

  22. TIC FUS

  23. Ramos lymphocytes 64 μg /90 μL*3=2.13 μg, EM ER, MRM Digestion overnight and clean up withstagetips, sampleverydirty, 100 μL of Urea 6M

  24. Ramos lymphocytes, EM ER RESULTS

  25. Ramos lymphocytes, EM ER RESULTS

  26. MRM Q-TRAP 5500, Ramos lymphocytes

  27. TIC COR1A

  28. MRM COR1A

  29. MRM Ramos Q-TRAP 5500

  30. MRM Ramos Q-TRAP 5500

  31. 100μg/140μl of 2%ACN 0.1% FA, 0.71*3μL=2.13ug. EM ER Q-TRAP 5500 STAGE TIPS, chondrocytes

  32. RESULTS EM ER FUS

  33. MRM PILOT Q-TRAP 5500, chondrocytes Theidentification of the 20 proteinswasnotimproved

  34. MRM CONCLUSIONS Q-TRAP 5500, METHOD FOR FUS CPSF5 UBP10 COR1A

  35. O81821 DP80 DT25 FINAL034

  36. CPSF5 COR 1A FUS UBP10

  37. FUS MRM

  38. FUS MRM

  39. UBP10 MRM

  40. UBP10 MRM

  41. UBP10 MRM

  42. CPSF5 MRM

  43. CPSF5 MRM

  44. CPSF5 MRM

  45. CCD18 EM ER • 50μg /70 μL*3=2.13 μg • Trap Acclaim Pep Map 100 nano viper 100µm*2cm, C18, 5 µm, 100A • 10 min*3 µl/min , 3µL injection

  46. CCD18 EM ER

  47. CCD18 MRM . Only two proteins were detected

  48. MCF7 EM ER 50μg /70 μL*4=2.8 μg Trap Acclaim Pep Map 100 nano viper 100µm*2cm, C18, 5 µm, 100A 10 min*3 µl/min , 4µL injection

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