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Home arrow Health arrow Analysis of Protein Post-Translational Modifications by Mass Spectrometry
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Bioinformatics

Generally, identification and localization of citrulline are performed using database search algorithms such as Mascot and SEQUEST, as discussed earlier. It should be noted that if the sample was digested with trypsin, the number of allowed missed cleavages should be greater than that used for a normal search.

As mentioned earlier, the detection of citrullinated peptides is complicated by the presence of deamidated asparagine and glutamine residues. Bennike et al. [89] suggest that the presence of missed cleavages on tryptic peptides can be used to increase confidence in assigning modified peptides. Using 24 biologically relevant citrullinated peptides (no C-terminal citrullination sites) and the unmodified counterpart peptides, they assessed the potential for missed cleavages (along with the retention time shift, as described earlier). The 48 peptides were digested with trypsin and analyzed by LC-MS/MS. In all cases, they found that the citrullinated peptides resulted in missed cleavage products, whereas the unmodified counterpart peptides were fully digested. Deamidation does not inhibit trypsin and would behave in the same way as the unmodified peptides. As discussed earlier, they then applied the knowledge that citrullination would result in missed cleavage from trypsin digestion to analyze synovial fluid.

De Ceuleneer et al. [116] describe a method whereby coeluting citrullinated and noncitrullinated peptides can be analyzed to determine the percentage of citrullination (“skewing”). Using the endoprotease Lys-C for the sample digestion will result in citrullinated and noncitrullinated peptides having the same sequence. This method is reliant on the coelution of citrullinated and non- citrullinated peptide, which has been shown to not always be the case by Bennike et al. [89]; however, for those peptides that do coelute, De Ceuleneer et al. [116] provide a template that can be used to determine the percentage “skewing” A large proportion of manual analysis is involved: theoretical isotope distributions need to be manually created for the citrullinated and noncitrul- linated forms, and the isotope pattern from the MS data must be manually extracted. With synthetic citrullinated and noncitrullinated peptides, De Ceuleneer et al. observed good linear fit between the calculated and measured percentages (R2 > 0.9). They subsequently calculated the skewing for several citrullinated peptides from synovial fluid.

 
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