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Although bottom-up proteomics using tryptic digests is widely used to locate post-translational modifications (PTM) in proteins, there are cases where the protein has several potential modification sites within a tryptic fragment and MS2 strategies fail to pinpoint the location. We report here a method using two proteolytic enzymes, trypsin and pepsin, in combination followed by tandem mass spectrometric analysis to provide fragments that allow one to locate the modification sites. We used this strategy to find a glycosylation site on bovine trypsin expressed in maize (TrypZean). Several glycans are present, and all are attached to a nonconsensus N-glycosylation site on the protein. 10.1021/ac1020722

The role of calcium and other cations is characterized in the events of the catalytic turnover cycle. Certain 3d-cations bind to the active site of PLA and some are Ks cofactor for substrate or mimic binding, although without measurable kcat. The kcat cofactor role of a cation is seen in terms of a catalytic mechanism, where the water nucleophile in the expanded coordination area of the cation could be in a favorable near-attack conformation.

In-source direct pyrolysis mass spectrometry is studied for rapid identification of polymers in industrial products. In this technique, polymers are pyrolyzed within the ion source of a mass spectrometer by a coiled filament designed for desorption chemical ionization/desorption electron ionization applications. Pyrolysis products are ionized by 70 eV electron impact, yielding highly reproducible mass spectra characteristic of the polymer composition. Using this technique and a commercial library editor, a comprehensive mass spectral library containing more than 150 polymer entries was developed. The impacts of experimental and polymer variables on the reproducibility of polymer mass spectra were evaluated. The technique was successfully applied to the analysis of polymers in industrial products, showing advantages of short analysis time, high confidence level, and low matrix interference. This study suggests that a robust standard polymer library, independent of laboratory and mass spectrometer type, may be developed. However, more rigorous interlaboratory tests are needed to achieve this goal.

The use of deuterium oxide as a mobile phase in the routine analysis of pharmaceutical compounds was investigated. The deuterium exchange of labile hydrogen atoms aids in structural confirmation and elucidation of unknown impurities and degradation products. Although deuterium oxide as a mobile phase does in some cases change the retention limes, the changes in retention times do not interfere with the analysis. A study of the high-performance liquid chromatography system shows that equilibration times for the deuterium-containing mobile phases are similar to equilibration times with changes of other mobile phases. The use of this technique in the analysis of pharmaceutical compounds and other small molecules is presented.