Dr. Lebrilla

Carlito Lebrilla; Evan Parker; Michael Xin Sun; Jincui Huang; Andres Guerrero

UC Davis, Davis, CA

Novel Aspect
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.

Preliminary Results/Abstract
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.

The N-glycosylation sites of bovine fetuin are an excellent example of why multiple proteases should be used to elucidate site-specific structures. Using the standard workflow of tryptic digestion, N-glycans on sites 99, 156, and 176 can be found in peptides of length 32, 15, and 28 respectively. When the potential for a single missed cleavage is taken into account peptides of length 49, 43, and 52 are possible. Using elastase as a proteolytic enzyme, peptides are much shorter, 4, 7, and 10 amino acids long at minimum and 11, 18, and 15 amino acids long with one missed cleavage. These small peptides are much easier to detect and have easier to interpret CID spectra. A further step we take is the use of nonspecific proteases; with nonspecific digestion peptides are observed from length 2 to 10 at all eight sites.As an additional example, horseradish peroxidase has eight glycosylation sites. Site 43, 216, and 228 are difficult to analyze by tryptic digestion due to the large peptide generated at site 43 and the fact that sites 216 and 218 share a tryptic peptide. While elastase could aid in the assignment of site 43, it actually complicates the assignment of site 216 and 228 further since the smallest peptide includes site 244 as well. Pronase performs excellently on this protein producing many peptides at each site from 2 to 10 amino acids long.