Stable isotopically labeled (SIL) tryptic peptides, cleavable SIL peptides, and a full-length SIL protein were compared for internal calibration (i.e., as internal calibrators) and external calibration (i.e., as internal standards) when quantifying three forms of unlabeled, human thyroglobulin (Tg) by bottom-up protein analysis. All SIL materials and human proteins were standardized by amino acid analysis to ensure traceability of measurements and allow confident assignment of accuracy. The three forms of human Tg quantified were (1) the primary reference material BCR457-a native protein purified from human thyroids, (2) a commercially available form also purified from human thyroids, and (3) a full-length recombinant form expressed and purified from a human embryonic kidney 293 cell-line. Collectively, the results unequivocally demonstrate the lack of commutability of tryptic and cleavable SIL peptides as internal calibrators across various bottom-up assays (i.e., denaturing/digestion conditions). Further, the results demonstrate the potential during external calibration for surrogate protein calibrators (i.e., recombinant proteins) to produce inaccurate concentration assignments of native protein analytes by bottom-up analysis due to variance in digestion efficiency, which is not alleviated by altering denaturation/digestion stringency and indicates why protein calibrators may not be commutable in bottom-up protein assays. These results have implications regarding the veracity of "absolute" protein concentration assignments by bottom-up assays using peptide calibrators, as well as protein calibrators, given that absolute accuracy was not universally observed. Nevertheless, these results support the use of recombinant SIL proteins as internal standards over SIL peptides due to their ability to better mimic the digestion of human-derived proteins and mitigate bias due to digestion-based matrix effects that were observed during external calibration.

We present a new time-dependent model of the interplanetary heavy ion environment and a new set of software based on this model to calculate energy deposit (LET) spectra and resulting single event upset rates.

SEUs in the CRRES MEP showed a dramatic increase during a solar flare, the influence of the flare varied widely among device types, and a GaAs RAM showed a different response to the proton belts than some Si RAMS. Corrections to the SEU rate to account for orbital dwell time emphasize the dramatic difference between the rate in the proton belts and the rate due to cosmic ray ions above the belts. In the case of one device, apparent total dose damage resulted in a large increase in upsets due to unreliable device operation.

Although bottom-up proteomics using tryptic digests is widely used to locate posttranslational 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.

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