Samples were digested using the In-gel Tryptic Digestion Kit (Thermo Scientific) according to the manufacturer’s instructions. The tryptic digest mixture was dried and resuspended in 50:50 acetonitrile:water, then mixed with an equal volume of α-cyano-4-hydroxycinnamic acid ionisation matrix (α-CHCA) and applied to a Bruker Daltonics UltrafleXtreme 2 mass spectrometer operated in positive ion reflection mode. The results were analysed using MASCOT Peptide Mass Fingerprint (Matrix Science), searching the NCBInr database with fixed carbamidomethyl modification and variable oxidation modification, and a peptide tolerance of ± 200 ppm.
Mascot peptide mass fingerprint
Manufactured by Matrix Science
The MASCOT Peptide Mass Fingerprint is a software tool used for the identification of proteins from mass spectrometric data. It compares the experimental mass spectra of peptides against theoretical mass spectra derived from protein sequence databases to determine the most likely protein match.
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Lab products found in correlation
2 protocols using mascot peptide mass fingerprint
1
In-Gel Tryptic Digestion and MALDI-TOF MS Analysis
2
Analytical Techniques for Biochemical Analysis
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Ammonia concentration was measured with the colorimetric method described by Taylor et al. (62 (link)). Nitrite was measured with the Griess reaction as described by Kartal et al. (63 (link)). Protein concentration was measured with the Bio-Rad protein assay based on the method of Bradford (64 (link)), using bovine serum albumin as standard. The identification of the isolated protein complex was confirmed by MALDI-TOF mass spectrometry (56 (link)). Each spectrum (900–4000 m/z) was analyzed using the Mascot Peptide Mass Fingerprint (Matrix Science) against the K. stuttgartiensis database, allowing methionine oxidation as variable modification, 0.2-Da peptide tolerance, and at most one trypsin miscleavage.
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