Mascot sequence matching software

Spots showing differential expression in brain and Neuro2a cells were analyzed at the central instrumental facility at the Delhi University South Campus and identified by Applied Biosystem 4800 plus MALDI TOF/TOF Analyzer (AB Sciex, USA) as previously described. For MALDI-TOF mass spectrometry, 1 µl of the digest was mixed with 2 µl of the matrix solution (α-cyano-4-hydroxycinnamic acid in 80% [vol/vol] acetonitrile and 0.1% [vol/vol] trifluoroacetic acid [TFA]), and 1 µl of this mixture was deposited onto the MALDI target. The spectrum was obtained in the mass range of 500 to 4,000 Da and was calibrated using a calibration mixture containing angiotensin I, Substance P, adrenocorticotropin(1–17) [ACTH(1–17)], ACTH(18–39), and somatostatin [17] (link).Spectra were analyzed with MASCOT sequence-matching software from Matrix Science. The peak list was searched against the taxonomy group Mus musculus at non-redundant protein sequence database of NCBI with 144127 sequence entries. Search parameters were as follows: trypsin digestion with one missed cleavage, variable modifications (oxidation of methionine and carbamidomethylation of cysteine), and the peptide mass tolerance of 50 ppm for precursor ion and mass tolerance of ±0.6 Da for fragment ion with +1 charge state.

SDS gels were cut and subjected to in-gel tryptic digestion to identify amino acid sequences, as carried out by the Ward Medic company (www.wardmedic.com). Tryptic peptide samples were analyzed for amino acid sequences using the Shimadzu Prominence nano HPLC system [Shimadzu] coupled with a 5600 TripleTOF mass spectrometer [Sciex]. Data from mass spectrometry analysis were interpreted to identify peptide sequences by using Mascot sequence matching software [Matrix Science] based on the UniProt database with p-value < 0.05.

Two hundred and fifty μl of serum PCA isolate (120 μg/ml) was added to the Sepharose conjugated CGB lectin beads and incubated for 90 min at room temperature with constant vortex. Unbound proteins were removed by centrifugation and the beads were washed as earlier described. The CGB lectin bound proteins were released by adding 25 μl of Laemmli buffer and incubated at 95°C for 5 min. Released peptides were resolved in 8% SDS-polyacrylamide gel (Biorad, Hercules, CA, USA). Peptide bands were visualized by silver staining. The developed gel was scanned using ImageScanner III (GE Healthcare, Little Chalfont, BU, UK) and image analysis was performed using GelAnalyzer software. Each of the peptide bands was excised and subjected to typical in-gel trypsin digestion [16 (link)]. The extracted peptides were analysed using Agilent 6540 mass spectrometer (Agilent, Santa Clara, CA, USA). Proteins were identified by subjecting the spectra to Mascot sequence matching software (Matrix Science) analysis using the Ludwig NR database.

Generated mass spectral data were analyzed using Mascot sequence matching software (Matrix Science). General search parameters used were: Enzyme, Trypsin; Maximum of 2 missed cleavages; Fixed modifications, Carbamidomethyl (C); Variable modifications, Oxidation (M), Deamidated (NQ), Phospho (STY), Acetyl (K); Peptide tolerance range ±100 ppm; and MS/MS tolerance range, ±0.6 Da. Data import filters used were Mascot Distiller (Matrix Science). Analyzed database-search results (.dat) from Mascot were uploaded onto Scaffold interface. Result files were categorized and named according to sample type or nature. Multidimensional protein identification technology (MuDPIT) experiment and condensation of loaded data were selected. Proteins were designated as hits only when there were at least 2 unique peptides matches and Protein Identification Probability value of >95%.
The quantitation of protein expressions by spectral counts for each identified proteins (defined as total spectral counts for all the peptides of a given protein) was carried out using Scaffold program (Proteome Software, version 4). Only proteins with spectral counts ≥5 and a ratio of spectral counts between two groups (patients and normal controls) of >2 or <0.5 were tested further by t-test and validation by immunohistochemistry.

The Coomassie-stained protein spots corresponding to those recognized by the above sera were manually excised and transferred to microcentrifuge tubes. These protein spots were analyzed using mass spectrometry analysis by First Base Laboratories Sdn Bhd, Malaysia. Protein samples were trypsin digested and peptides extracted according to standard techniques. Peptides were analyzed by matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometer using a 4800 Proteomics Analyzer. Spectra were analyzed to identify proteins of interest using Mascot sequence matching software (Matrix Science) with Ludwig NR Database and taxonomy set to other metazoa.

In preparation for liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Wischgoll et al., 2009), protein samples from mussels receiving NLHS were resolved in 5% SDS polyacrylamide gels by electrophoresis, stained with Biosafe Coomassie (BioRad Laboratories) and destained. Gel slices containing PvHsp70-1 and PvHsp70-2 were excised and freeze-dried overnight. Proteins were then digested with trypsin and the extracted peptides [24 (link)] were loaded onto a C18 column 300SB, 3.5 μm column (Agilent Technologies, USA) and separated with a linear gradient of water/acetonitrile/0.1% formic acid (v/v). The peptides were analyzed by electrospray ionization mass spectrometry with a Shimadzu Prominence Nano HPLC system (Shimadzu, Japan) coupled to a 5600 TripleTOF mass spectrometer (AB Sciex, USA). Protein identification was performed with Mascot sequence matching software (Matrix Science, USA) and the Ludwig NR database.