Paragon algorithm

Protein bands were excised from the polyacrylamide gels and subjected to reduction (10 mM dithiothreitol), alkylation (50 mM iodoacetamide), and overnight ingel digestion with sequencing grade trypsin (Sigma), in 50 mM ammonium bicarbonate at 37 °C. Tryptic peptides were extracted with 50% acetonitrile containing 1% TFA, and analyzed by MALDI-TOF-TOF on an AB4800-Plus Proteomics Analyzer (Applied Biosystems). Peptides were mixed with an equal volume of saturated α-cyanohydroxycinnamic acid (in 50% acetonitrile, 0.1% TFA), and spotted (1 μL) onto an Opti-TOF 384-well plate, dried, and analyzed in positive reflector mode. TOF spectra were acquired using 500 shots at a laser intensity of 3000. Selection of the ten most intense precursor ions was done automatically and their TOF/TOF fragmentation spectra were acquired using 500 shots at a laser intensity of 3900. External calibration in each run was performed with CalMix ® standards (ABSciex) spotted onto the same plate. Resulting spectra were searched against the UniProt/SwissProt database for Serpentes (20150217) using ProteinPilot ® v.4 and the Paragon ® algorithm (ABSciex) at ≥ 95% confidence, or, in few cases, manually interpreted and the deduced sequences searched using BLAST (http://blast.ncbi.nlm.nih.gov) for protein family assignment by similarity.

Protein bands were excised from gels, destained with 50% acetonitrile in 25 mM ammonium bicarbonate, and then subjected to reduction (10 mM dithiothreitol), alkylation (50 mM iodoacetamide), and overnight in-gel digestion with sequencing grade trypsin (Sigma), in 50 mM ammonium bicarbonate at 37°C. The resulting tryptic peptides were extracted with 50% acetonitrile containing 1% trifluoroacetic acid (TFA), and analyzed by MALDI-TOF-TOF on an AB4800-Plus Proteomics Analyzer (Applied Biosystems), under conditions previously described (Lomonte et al., 2014) . In each run, CalMix ® standards (ABSciex) spotted onto the same plate were used as external calibrants. Resulting spectra were searched against the UniProt/SwissProt database using ProteinPilot ® v.4 and the Paragon ® algorithm (ABSciex) for protein identification at ≥ 95% score confidence, or manually interpreted. Few peptide sequences with lower confidence scores were manually searched using BLAST (http://blast.ncbi.nlm.nih.gov)
for protein similarity and assignment to protein families.

From the polyacrylamide gels protein bands were excised and subjected to reduction (10 mM dithiothreitol), alkylation (50 mM iodoacetamide), and overnight in-gel digestion with sequencing grade trypsin (Sigma), in 50 mM ammonium bicarbonate at 37 °C. Tryptic peptides were extracted with 50% acetonitrile containing 1% TFA, and analyzed by MALDI-TOF-TOF on an AB4800-Plus Proteomics Analyzer (Applied Biosystems). Digested and extracted peptides were mixed with an equal volume of saturated α-cyano-hydroxycinnamic acid (in 50% acetonitrile, 0.1% TFA), and spotted (1 μL) onto an Opti-TOF 384-well plate, dried, and analyzed in positive reflector mode. TOF spectra were acquired using 500 shots at a laser intensity of 3000. TOF/TOF fragmentation spectra were acquired using 500 shots at a laser intensity of 3900 for the automatically selected ten most intense precursor ions. CalMix ® standards (ABSciex) spotted onto the same plate were used for external calibration in each run. Resulting spectra were searched against the UniProt/SwissProt database for Serpentes (20150217) using ProteinPilot ® v.4 and the Paragon ® algorithm (ABSciex) at ≥ 95% confidence, or, in few cases, manually interpreted, and the deduced sequences searched using BLAST (http://blast.ncbi.nlm.nih.gov) for assignment of protein family by similarity.