The GlycoFilter: A Simple and Comprehensive Sample Preparation Platform for Proteomics, N-Glycomics and Glycosylation Site Assignment

Abstract

Current strategies to study N-glycoproteins in complex samples are often discrete, focusing on either N-glycans or N-glycosites enriched by sugar-based techniques. In this study we report a simple and rapid sample preparation platform, the GlycoFilter, which allows a comprehensive characterization of N-glycans, N-glycosites, and proteins in a single workflow. Both PNGase F catalyzed de-N-glycosylation and trypsin digestions are accelerated by microwave irradiation and performed sequentially in a single spin filter. Both N-glycans and peptides (including de-N-glycosylated peptides) are separately collected by filtration. The condition to effectively collect complex and heterogeneous N-glycans was established on model glycoproteins, bovine ribonuclease B, bovine fetuin, and human serum IgG. With this platform, the N-glycome, Nglycoproteome and proteome of human urine and plasma were characterized. Overall, a total of 865 and 295 Nglycosites were identified from three pairs of urine and plasma samples, respectively. Many sites were defined unambiguously as partially occupied by the detection of their nonsugar-modified peptides (128 from urine and 61 from plasma), demonstrating that partial occupancy of N-glycosylation occurs frequently. Given the likely high prevalence and variability of partial occupancy, glycoprotein quantification based exclusively on deglycosylated peptides may lead to inaccurate quantification. Molecular

Absolute Quantitation of Glycosylation Site Occupancy Using Isotopically Labeled Standards and LC-MS

“…As a demonstration of the method, the partially occupied glycosylation site of bovine fetuin at Asn-158 was studied [16-17]. As a first step, we verified that no additional PTMs were present on this peptide.…”

18O-incorporated De-N-glycosylated Site Identification: A Comparison of Three Search Engines

“…If a specific motif was identified in the deglycosylated form and also identified in either of the other two forms, this indicated that that motif was partially glycosylated [8]. When all motifs were compared, evident differences were observed among the results from three search engines (Figure 1a -c).…”

“…The depletion of albumin in urine was performed according to a published one-step protocol [15], and the protein concentration was measured by the Bradford assay in triplicate. Approximately 250 µg of depleted urinary proteins from each donor were further processed according to the GlycoFilter platform to obtain released N-glycans and tryptic peptides [8], respectively. PNGase F catalyzed de-N-glycosylation was carried out in a H 2 18 O buffer via a 20-min domestic microwave protocol [16].…”

“…First, the removal of complex and heterogeneous sugar residues enables the detection, characterization, and even quantification of deglycopeptides by standard proteomic methodologies [7]. Second, the stripping off of sugar-moieties allows the detection of both deglycopeptides and their counterpart unmodified (non-glycosylated) peptides simultaneously, which are necessary to identify partially glycosylated sites unambiguously [8]. Third, the sugar residues can be employed to enrich for glycoproteins or glycopeptides using hydrazide-chemistry or lectins [7,9].…”

“…Unfortunately, this product is identical to those derived from spontaneous chemical deamidation [11], which may occur in vivo or in vitro. Researchers have shown that performing the enzymatic reaction in an H 2 18 O environment is effective in differentiating enzymatic conversion (Asn to 18 O-Asp, +2.9883 Da) from chemical deamidation [8,9]. Identifying 18 O-incorporated deglycopeptides usually involves searching MS/MS spectra against an appropriate protein database using various search engines such as Mascot [12], ProteinPilot [13], and Sequest [14].…”