Simultaneous Protein Identification And Quantification In MS/MS Spectrum&the Designing And Testing Of¹⁸O Labeling Quantitative Formula

The relative quantification and identification of proteins by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and MS/MS are very important in protein research and are usually conducted separately. Chemical N-terminal derivatization with4-sulphophenyl isothiocyanate (SPITC) facilitates de novo sequencing analysis and accurate protein identification, while18O labeling is simple, specific and widely applicable among the isotopic labeling methods used for relative quantification. Precursor peaks in MS/MS spectrum could provide accurate quantitative information which is usually acquired from MS spectrum, enabling protein identification and quantification in a single MS/MS spectrum. Next, this method was applied to native peptides isolated from spider venoms. As expected, the de novo sequencing results of each peptide matched with the known sequence precisely and the measured quantitative ratio of each peptide corresponded well with the theoretical ratio. The utility of the method has been confirmed in both simple and complex sample determinations, such as Lycosasingoriensis spider venom with no genomic information. These approaches may therefore pave the way for successful exploration of peptides/proteins from species with unknown genomes. Among the various isotopic labeling techniques for relative quantification,18O labeling is simple, specific, and cost-effective for a wide range of analyses. But two disadvantages were exposed during experiments:"interference of natural isotope peaks" and "loss of18O atom". The fomer effect could not be dispelled or beignored, and causing inaccuracy. The latter effect was due to18O atomic motion. Reducing the time bettwen labeling and mass spectrum dentification, proper pH and temperature of storage after labeling might decrease the loss of18O atom. In this article, we focus no longer on how to decrease these effects but on how these two affection make quantification inaccurate.18O labeling quantitative formula was decuced and tested on sample with high natural isotope peaks and loss of18O atom. And the experimental results after correction of this formula matched well with theoretical data.