1-DE-LC/ESI-MS
Processed slices of the 1D gels of Pellet, Supprot and Sephadex were analyzed by nano-LC
ESI-MS/MS. Peptides were separated on an Agilent 1100 series chromatography unit (Agilent
Technologies, Palo Alto, CA, USA). Samples were dissolved in 12 µl 0.1% formic acid, 3%
ACN and centrifuged for 10 min at 13800 g. From the resulting supernatant 6 µl were loaded
onto a trap column Zorbax 300SB – C18, 5 x 0.3 mm (Agilent Technologies) and washed 5
min with a flow rate of 0.02 ml/min and solution A (3% ACN, 0.1% formic acid). The
peptides were fractionated with a Zorbax 300SB-C18, 3.5 µm, (75 µm I.D. x 150 mm, Agilent
Technologies) at a flow rate of 0.3 µl/min. The gradient was built up with ACN/formic acid
(3%/0.1%) as solvent A and ACN/formic acid (99.9%/0.1%) as solvent B. The elution
gradient was 3–15% B for 3 min, 15–45% B for 42 min, 45–90% B for 5 min.
The column outlet was coupled online to an LC/MSD Trap XCT mass spectrometer (Agilent
Technologies). Precursor masses with m/z ratio of 200 – 2200 were allowed. MASCOT
(Version 2.1) MS/MS Ion Search was used to search against the HP26695 TIGR database
with the following criteria: A peptide mass tolerance of ± 0.4 Da and a fragment mass
tolerance of ± 0.4 Da were accepted. A maximum of 1 missed cleavage was allowed and the
same variable modifications as defined above.
For ESI-MS/MS data analysis, the ions score cut-off was 20 and proteins were classified as
identified, if either at least two peptides with a MASCOT-score above the statistically
relevant threshold (p<0.05) were found or, if only 1 peptide achieved the required MASCOT
score, that at least 4 consecutive y- or b-ions with a significant signal to noise ratio were
determined.