Spots were robotically excised from colloidal Coomassie-stained (Bio-Safe; Bio-Rad,
Hercules, CA, USA) preparative gels loaded with 1.5 mg protein (pooled from LCR and
HCR samples) and proteins identified by MADLI-MS/MS analysis of in-gel tryptic digests,
as described previously {Burniston 2009a}. Peak list were searched against the Swiss-Prot
database (57.1) restricted to ‘Rattus’ (7347 sequences) using a locally implemented
Mascot (www.matrixscience.com) server (version 2.2.03). The enzyme specificity was
trypsin allowing 1 missed cleavage, carbamidomethyl modification of cysteine (fixed),
oxidation of methionine (variable) and an m/z error of ± 0.5 Da. Identification
was accepted based on a significant Mowse score; the threshold (P<0.05) was 51 using
the described database constraints. Up to 3 confirmatory MS/MS spectra were automatically
acquired from digests with Mowse scores <120. Selection of MS/MS fragment ion spectra
(average m/z) was restricted to 42 ions over 6 segments encompassing 5 – 95 % of
the precursor ion m/z. MS/MS ions lists were searched against the Swiss-Prot database
(tolerance ± 0.5 Da parent, ± 0.8 Da fragments) using the constraints described
for peptide mass fingerprinting.