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Pipeline publication

[…] MALDI-TOF Voyager-DE™ PRO (Applied Biosystems) using a delayed ion extraction and ion mirror reflector mass spectrometer. The instrument settings were: reflector mode with positive polarity, 100 nanosecond delay extraction time, 70% to 80% grid voltage and 20,000 V accelerating voltage. Laser shots at 500 per spectrum were used to acquire one spectrum with a mass range from 700 to 4,000 Da. External calibration was carried out using the Proteomix–Peptide calibration Mix4 (LaserBio Labs). Spectra were accumulated manually from different acquisitions to improve resolution and the signal-to-noise ratio., The tools used to identify proteins from peptide mass fingerprinting data were Aldente and FindMod [,], which can be found on the Expasy server []. By looking over differences between experimentally determined and theoretical peptide masses from a specified protein, FindMod permits one to discover PTMs and to make predictions as to what amino acid in the peptide is likely to carry the modification. Several possibilities were often suggested that stand within the selected mass tolerance, but most of them could be eliminated using a manual spectrum recalibration. The peptides were generated by trypsin that cleaves proteins at the C-terminal side of K or R. The number of missed cleavages allowed was set to 1 for Aldente and was set up to 3 for FindMod analysis. Several chemical modifications occurring during the separation process were taken into account in Aldente and FindMod analysis: carboxyamidomethyl cysteine due to the action of iodoacetamide on cysteine residues, propionamide cysteine that is an acrylamide adduct to cysteine, and methionine sulfoxide linked to the presence of ammonium persulfate in the gel., After the identification of immunoreactive proteins with the Aldente program, the corresponding spectra were further examined in order to detect the presence of several types of PTM of discrete mass. The FindMod and FindPept programs (Expasy server []) were used for looking at mass differences between experimentally determined peptide masses and theoretical peptide masses. When a mass difference corresponding to a known PTM was observed, rules were applied that examine the sequence of the peptide of interest and make predictions as to which amino acid in the peptide was likely to carry the modification. These rules are included either in the FindMod and FindPept programs or in the various tools and software for PTMs found on the Expasy server [] (for instance, NetPhos or NetAcet)., In our study, a particular attention was paid to citrullination, a PTM occurring on arginine residues. Several rules were applied: for one citrullinated arginine, the peptide theoretical mass increase is 0.98 Da and the modified peptide, losing one amino group, becomes more acidic []; citrullinated arginine residues are not likely to be cleaved by trypsin, so that a minimum number of one missed cleavage must be specified and a peptide that includes a C-terminal citrullinated arginine must be rejected; and in a biological sample, only a fraction of a given protein may be citrullinated at a specific site. Because of the several PTMs occurring on a given protein, this protein was gen […]

Pipeline specifications

Software tools FindMod, NetPhos, NetAcet