The identification of spots from 2-DE gels was described in detail [28]. After digestion the
peptides were dissolved in 1.3 µl 33% ACN, 0.1% TFA; 0.25 µl of the solution was mixed
with 0.5 µl 5 mg/ml alpha-cyano-4-hydroxycinnamic acid (Aldrich 1302B1, recrystallized
and stored at -20°C), 50% ACN, 0.3% TFA and spotted onto the MALDI steel target. Protein
spots that failed identification were further purified with ZipTipµ-C18 (Millipore, Schwalbach,
Germany) according to the instructions of the manufacturer. The samples were analyzed in
the MS mode for generation of peptide mass fingerprints (PMF) with a 4700 Proteomics
Analyzer (Applied Biosystems, Darmstadt, Germany) [28]. Concomitantly, the peptides of the
three most intense peaks were fragmented in the TOF/TOF mode to complement the protein
identification by PMF. MS spectra were calibrated internally by trypsin fragments and known
contaminants containing matrix, trypsin and keratin peptide masses (suppl. Material Table 1)
were excluded by an exclusion list. Protein identification was performed using the GPS
Explorer Software 3.5. and was achieved by database comparison against the H. pylori 26695
subset of the NCBI database using the search algorithm MASCOT 1.9. A peptide tolerance of
30 ppm, a MS/MS tolerance of 0.3 Da and one missed cleavage were allowed. Moreover, the
variable modifications N-terminal acetylation, Met-oxidation, propionamide (C) and
pyroglutamic-acid on N-terminal Gln were allowed. An exclusion list was generated
(supplemental material Table2 excel) using MS-Screener [32], whereby mass values which
occurred with a frequency of more than 5% and those which did not fulfil the half decimal
place rule were excluded.
Proteins were considered as identified, if (i) the sequence coverage was >30%, or (ii) the
sequence coverage was > 10% and at least one MS/MS spectrum confirms identification and
the MascotScore is >44 with p <0.05, or (iii) in some rare exceptions the first two criteria
were not fulfilled, but the spot under investigation was within a series of identified spots or
the mass loss rules verified an MS/MS assignment [33].