The determination of site-specific glycosylation in proteins remains a difficult task. Two methods currently in use employ specific proteases such as trypsin or a non-specific cocktail of proteases. Trypsin yields large well-defined glycopeptides. These large glycopeptides can be difficult to analyze by tandem MS. Additionally, glycopeptides may be produced containing two glycosylation sites. Nonspecific proteases yield smaller glycopeptides that vary in length and are more difficult to predict. Presented here is a comparison between the trypsin approach and that using nonspecific digestion. A direct comparison is now possible because we have developed software to analyze both tryptic and non-tryptic glycopeptides. We find that the nonspecific protease method often has higher sensitivity and more complete coverage of site-specific glycan heterogeneity.
In-gel digestions were used to produce the glycopeptides. Briefly, 10 μg of a target protein was reduced with dithiothreitol and alkylated with iodoacetamide prior to running on SDS-PAGE. Gel bands were cut, destained, and dried in a speed-vac. To the dried gels, an appropriate amount of trypsin or pronase was added in ammonium bicarbonate buffer. After digesting overnight the glycopeptides were extracted. Samples were dried and resuspended in 20 μL prior to analysis on an Agilent HPLC-Chip/TOF MS. The resulting data was analyzed with in-house software utilizing accurate mass and fragment scoring to confirm identification.
A systematic comparison was performed involving several proteins. Proteins were selected due to their abundance in human serum, applications to biotechnology, or complexity. Included in this group are plant glycoproteins, which often contain α(1-3) linked fucose on the core glucoseamine. The lack of biochemical tools to cleave glycans with this linkage makes plant glycoproteins difficult to characterize by methods requiring enzymatic glycan release. For brevity, only the results of immunoglobulin M and α-2-macroglobulin from human serum are provided.In IgM, tryptic digestion yielded 17 glycan compositions at three sites, while nonspecific digestion yielded 26 compositions across all five potential glycosylation sites. Of the five sites, two were not observed with tryptic analysis because they fell within the same peptide. When IgM was digested and analyzed using the nonspecific digestion, more compositions were monitored and each result was achieved with higher confidence due to the higher mass accuracy afforded by the lower mass peptides and the presence of peptide-specific scoring ions.In α-2-macroglobulin, tryptic digestion yielded six glycan compositions at one site while nonspecific digestion yielded 28 glycan compositions at all eight sites. This protein is an even more striking example of the difference between these methods. Of the eight glycosylation sites half were inaccessible to tryptic analysis due to the fact that several sites shared a tryptic peptide. Only the peptide bearing site1424N was detected. Glycopeptides corresponding to the other sites were not observed, possibly due to their large sizes. The shorter peptides derived from nonspecific digestion afforded greater sensitivity and likely aided the search due to the reduced probability of unanticipated secondary posttranslational modifications.