Carlito Lebrilla; Evan Parker; Michael Xin Sun; Jincui Huang; Andres Guerrero
UC Davis, Davis, CA
Software allows the use of any enzymatic system for the analysis of glycopeptides by LCMS thereby allowing target-oriented glycopeptide analysis.
Site-specific analysis of protein glycosylation is an active and evolving field of research. Successful application of this analysis with respect to bio-activity is hampered by inconsistent analysis, difficult to repeat results, and extraordinarily low throughput. We have developed a workflow that counteracts several of the difficulties often encountered when assigning glycosylation site-structure pairs. Difficulties often found include peptides too large for accurate mass determination, hard to confirm structures due to multiple sites, and enzymatic resistance to the proteolysis. Our method avoids these problems by using a host of site-specific and nonspecific proteases to generate glycopeptides depending on the specific need. Using our own software platform we are able to analyze all of the data in a directly comparable manner.
Both in-gel and in-solution digestions are used, although in-gel digestion provides sensitivity advantages. Shortly, 10 ?g of a target protein is reduced and alkylated prior to running on SDS-PAGE. Gel bands are cut, destained, and dried in a speed-vac prior to digestion with trypsin, pronase, or elastase in ammonium bicarbonate buffer. After digesting overnight the glycopeptides are extracted. Samples were dried and resuspended in 20 ?L prior to analysis on an Agilent HPLC-Chip/TOF MS. The resulting data can be exported to MGF and analyzed with in-house software utilizing accurate mass and fragment scoring to confirm identification.
As a general example of the differences in digestion procedures, bovine fetuin and horseradish peroxidase serve as useful examples of potential pitfalls and benefits of each method. Horseradish perodidase is especially interesting because biochemical tools to cleave glycans in the presence of ?(1-3) linked fucose on the core glucoseamine are still new and difficult to obtain.