In-gel trypsin digestion was carried out as previously described . A 0.4 μl aliquot of the concentrated tryptic peptide mixture in 0.1% trifluoroacetic acid (TFA) was mixed find more with 0.4 μl of α-cyano-4-hydroxycinnamic acid (CHCA) matrix solution (5 mg/ml CHCA in 50% ACN/0.1% TFA) and spotted onto a freshly cleaned target plate. After air drying, the crystallized spots were analyzed on the Applied Biosystems 4700 Proteomics Analyzer MALDI-TOF/TOF (Applied Biosystems, Framingham, MA, USA). MS calibration was automatically performed by a peptide standard Kit (Applied Biosystems) containing des-Arg1-bradykinin (m/z 904), Angiotensin I (m/z 1296.6851), Glu1-fibrinopeptide B (m/z 1570.6774), Adrenocorticotropic hormone (ACTH)
(1-17, m/z 2903.0867), ACTH (18-39, m/z 2465.1989), and ACTH (7-38, m/z 3657.9294) and MS/MS calibration was performed by the MS/MS fragment peaks of Glu1-fibrinopeptide B. All MS mass spectra were recorded in the reflector positive mode using a laser operated at a 200 Hz repetition rate with wavelength of 355 nm. The accelerated
voltage was operated at 2 kV. The MS/MS mass spectra were acquired by the data dependent acquisition method with the 10 strongest precursors selected from one MS scan. All MS and MS/MS spectra were obtained by accumulation of at least 1000 and 3000 laser shots, respectively. Neither baseline subtraction nor smoothing was applied Epoxomicin manufacturer to recorded spectra. MS and MS/MS data were analyzed and peak lists were generated using GPS Explorer 3.5 (Applied Biosystems). MS peaks were selected between 700 and 3500 Da and filtered with a BLZ945 purchase signal to noise ratio greater than 20. A peak intensity filter was used with no more than 50 peaks per 200 Da. Tryptophan synthase MS/MS peaks were selected based on a signal to noise ratio greater than 10 over a mass range of 60 Da to 20 Da below the precursor mass. MS and MS/MS data were analyzed using MASCOT™ 2.0 search engine (Matrix Science, London, UK) to search against the C. themocellum protein
sequence database downloaded from NCBI database on December 01 2008. Searching parameters were as follows: trypsin digestion with one missed cleavage, variable modifications (oxidation of methionine and carbamidomethylation of cysteine), and the mass tolerance of precursor ion and fragment ion at 0.2 Da for +1 charged ions. For all proteins successfully identified by Peptide Mass Fingerprint and/or MS/MS, Mascot score greater than 53 (the default MASCOT threshold for such searches) was accepted as significant (p value < 0.05). The false positive rate was estimated based on reverse database search. The false positive rate = peptide fragment numbers detected in reverse database search/(peptide fragment numbers in forward database search+ peptide fragment numbers in reverse database search) × 100%. Acknowledgements The authors wish to acknowledge the kind assistance of Dr. Xiu-yun Tian for electrophoresis during the course of this study.