The identification of the major protein spots from two-dimensional gels was obtained by mass spectrometry. Coomassie blue-stained spots were obtained from at least two different bidimensional gels from two different separately treated preparations. The spots were cut into small blocks with a sterile scalpel blade. The blocks were rinsed, then reduced and alkylated with iodoacetamide, and incubated overnight at 37°C in a microtube with 12.5 ng/|xl trypsin (sequencing grade; Roche, Meillan, France) in 25 mM NH4HCO3 as previously described.
The tryptic fragments were extracted, dried, reconstituted with 0.1% formic acid, and sonicated for 10 min.
Tryptic peptides were analyzed either directly by MALDI (M@LDI-L/R; Waters Micromass, Manchester, UK) or sequenced by liquid chromatography coupled to tandem Mass Spectrometry (nano-LC-MS/MS) (Q-TOF-Global equipped with a nano-ESI source; Waters Micromass) in automatic mode. The peptides were loaded on a C18 column (Atlantis dC18, 3 |xm X 75 |xm X 150 mm, Nano Ease; Waters Micromass) and eluted with 5-60% linear gradient at a flow rate of 180 nl/min for 30 min (buffer A, water:acetonitrile 98:2 [v/v] 0.1% formic acid; and buffer B, water: acetonitrile 20:80 [v/v], 0.1% formic acid). The peptide masses and sequences obtained were either matched automatically to proteins in a non-redundant database (NCBI) using the Mascot program or de novo sequenced using the ProteinLynx Global Server program (Waters Micromass) and blasted manually against the current databases.
All reagents were of the best available grade (Sigma). Low molecular weight standards for electrophoresis were from Amersham-Pharmacia Biotech AB and Biorad (Ivry sur Seine, France).