Analytical performance of immobilized pronase for glycopeptide footprinting and implications for surpassing reductionist glycoproteomics

Abstract

A fully developed understanding of protein glycosylation requires characterization of the modifying oligosaccharides, elucidation of their covalent attachment sites, and determination of the glycan heterogeneity at specific sites. Considering the complexity inherent to protein glycosylation, establishing these features for even a single protein can present an imposing challenge. To meet the demands of glycoproteomics, the capability to screen far more complex systems of glycosylated proteins must be developed. Although the proteome wide examination of carbohydrate modification has become an area of keen interest, the intricacy of protein glycosylation has frustrated the progress of large-scale, systems oriented research on site-specific protein-glycan relationships. Indeed, the analytical obstacles in this area have been more instrumental in shaping the current glycoproteomic paradigm than have the diverse functional roles and ubiquitous nature of glycans. This report describes the ongoing development and analytically salient features of bead immobilized pronase for glycosylation site footprinting. The present work bears on the ultimate goal of providing analytical tools capable of addressing the diversity of protein glycosylation in a more comprehensive and efficient manner. In particular, this approach has been assessed with respect to reproducibility, sensitivity, and tolerance to sample complexity. The efficiency of pronase immobilization, attainable pronase loading density, and the corresponding effects on glycoprotein digestion rate were also evaluated. In addition to being highly reproducible, the immobilized enzymes retained a high degree of proteolytic activity after repeat usage for up to 6 weeks. This method also afforded a low level of chemical background and provided favorable levels of sensitivity with respect to traditional glycoproteomic strategies. Thus, the application of immobilized pronase shows potential to contribute to the advancement of more comprehensive glycoproteomic research methods that are capable of providing site-specific glycosylation and microheterogeneity information across many proteins.

Figure 1

nESI-FTICR mass spectra of 24 h RNase B (upper panel) and κ-CN (lower panel) pronase bead digests performed using the same bead preparations at several intervals over the course of six weeks. Glycopeptide signals are labeled with closed triangles and traced with arrows to the spectra below.

Figure 2

nESI-FTICR mass spectra of 10 μg, 1 μg, and 100 ng RNase B digests. Glycopeptide signals are labeled with closed triangles. Each trace represents the summation of 10 MS scans.

Figure 3

nESI-FTICR mass spectra of CEA, HAT, and BF digests. Glycopeptide signals are labeled with closed triangles and correspond to the glycopeptide compositions listed in Supplementary Tables 1 through 3.

Figure 4

Mean coupling efficiencies of differing masses of PE to varying amounts of S4B beads. Error bars represent the standard deviation of four coupling reactions. The amount of successfully coupled PE is expressed in both relative (upper panel) and absolute (lower panel) terms.

Figure 5

nESI-FTICR mass spectra of immobilized pronase digests of RNase B with 30 minutes of incubation (upper panel) and immobilized pronase digests of κ-CN with 120 minutes of incubation (lower panel). The digests using 1 mg, 5 mg, or 10 mg of PE are shown. Well known glycopeptides are labeled with closed triangles.

Figure 6

nESI-FTICR-MS analysis of a 1:1:1:1 (by mass) mixture of RNase B, κ-CN, BF, and the nonglycosylated protein BSA. Glycopeptide signals are labeled with closed triangles and correspond to the glycopeptide compositions listed in Supplementary Table 4.