The differential protein band at about 85 kDa was excised from the Coomassie blue-stained gel, and in-gel trypsin digestion was performed as previously described [21 (link)]. Briefly, the gel was destained and reduced, followed by alkylation and digestion. C18 ZipTips (Millipore Corp., Billerica, MA) was used to desalt the tryptic digested peptides. The desalted peptides were subjected to MALDI-TOF/TOF MS (Ultraflex-III, Bruker Daltonics, Bremen, Germany). The MS and MS/MS spectra were analyzed with the FlexAnalysis program (version 3.0, Bruker Daltonics) with default parameters. The matched peptides of the MS spectrum were searched via the MASCOT search engine for the protein identity using the peptide mass fingerprinting (PMF) approach and the MS/MS ion search approach. One missed cleavage in trypsin digestion was allowed among the search parameters. Phosphorylation of serine/threonine/tyrosine, methionine partial oxidation and iodoacetamide modification of cysteine residues were selected. The error tolerance values were 50 ppm and 0.1 Da, respectively, for the parent peptides and MS/MS ion masses.
Flexanalysis program
Manufactured by Bruker
FlexAnalysis is a software program developed by Bruker for the analysis of data from various analytical instruments. It provides a comprehensive suite of tools for data processing, visualization, and interpretation, enabling users to effectively analyze and interpret their experimental results.
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2 protocols using flexanalysis program
1
Protein Identification via Mass Spectrometry
2
MALDI-TOF MS Analysis of Biomolecules
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A sample and NSSA matrix (α-cyano-4-hydroxycoric acid in an aqueous solution of acetonitrile and trifluoroacetic acid) in 1:1 v/v were mixed and applied to the target surface (AnchorChip MALDI Target, Bruker Daltonics GmbH, Fällanden, Switzerland). The sample was dried and analyzed by MALDI-TOF MS in a Microflex LRF mass spectrometer (Bruker Daltonics GmbH) in a linear regimen. The produced mass spectra were analyzed with the FlexControl and FlexAnalysis software. Detectable masses ranged between 2 and 6 kDa. An individual mass spectrum was obtained by 40 laser pulses (60 Hz). The total mass spectrum obtained at 10 target points (400 laser pulses) was used for analysis. The laser power depended on the laser status to achieve optimal resolution and signal intensity. 19 Mass spectrometry strains analysis. Sample preparation was made as described by Hummel 20 . Protein extraction was carried out according to the protocol proposed by Bruker (Bruker Daltonics GmbH). The method of direct application of the culture to the target plate was also used 2 . The results were summarized using the FlexAnalysis program (Bruker Daltonics GmbH).
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