DATA ANALYSIS PROTOCOL FOR PROTEIN IDENTIFICATION

MALDI data acquisition

• To verify proteolysis, use one 1ul for MALDI TOF analysis.
• Dilute 1ul in 10 ul 0.1 %Formic acid. Desalt using ZipTip uC18, extract peptides with 50% saturated CHCA matrix in 60% Acetonitrile, 0.1% Formic acid.
• Spot 1ul on MALDI target, analyze in 4700 proteomics analyzer.

ESI MS data acquisition

• NanoHPLC (Eksigent 2D)
• Columns:
• For a single gradient run prepare a 10 cm long nanoRP HPLC column (100um ID)
• For 2D HPLC (MudPIT):  Prepare a 10 cm long 2D Column: 8 cm with RP, 1.5 cm with SCX, 0.5 cm RP column.
• Solvents:
• 0,1% formic acid
• 0.1% formic acid in Acetonitrile
• Single gradient run: 10% B in B+A for 5 min, 10 to 80% B in B+A in 75 min, and 90% B for 5 min.
• ESI MS analysis
• Mass spectrometer: LTQ FT.
• Data will be acquired on profile mode on the FT, with data scan dependence (6 MS/MS, on the IT). Exclusion list will be used.

MudPIT data acquisition

• NanoHPLC (Eksigent 2D). Using either direct injection to the MS, or after prefractionation of sample on an Eksispotter nano fraction collector
• Solvents:
  • 500 mM Ammonium acetate
  • 0,1% formic acid
  • 0.1% formic acid in Acetonitrile
  • 2D HPLC gradients: Elution from SCX using 12-20 steps (0 to 300 mM Ammonium acetate), each followed by a nanoHPLC MSMS as described above.
• Mass spectrometer: LTQ FT with same settings as described above

Data analysis

• MALDI data are used for peptide mass fingerprint. This data  will be analyzed using the MASCOT search engine, running on a local machine.
• ESI MSMS data will be analyzed using Sequest search program running on a 8 node dual processor dedicated computer cluster. Post processing can be done using either Bioworks browser, the set of applications from the Yates lab (DTAselect, contrast) or theTras Proteomics Pipeline (Institute for Systems Biology)