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, N-glycoproteome and proteome of human urine and plasma were characterized. Overall, a total of 865 and 295 N-glycosites 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.
Highlights
From the ‡Department of Urology and The Proteomics Center, Boston Children’s Hospital and Harvard Medical School, Boston, Massachusetts 02115
The extracted glycoproteins or glycopeptides are subjected to de-N-glycosylation, and the deglycosylated peptides are sequenced by liquid chromatography-tandem mass spectrometry (MS) (LC-MS/MS) to characterize the previously glycosylated sites with a standard bottom-up proteomic approach [10]
The GlycoFilter platform was evaluated in three distinct aspects: [1] the separation efficiency of released N-glycans by filtration, as filtration is not an established method to separate glycans from proteins; [2] the general impact on proteomic performance of complex biological samples after PNGase F catalyzed deN-glycosylation; and [3] the identification of N-glycosites and partially occupied (PO) N-glycosites
Summary
A glycosite may be partially occupied (PO), a state in which both the glycosylated (sugar-modified asparagine) and nonglycosylated (nonsugar-modified asparagine) forms coexist. The biological implications of partial occupancy in N-glycosylation are not well understood, to date, there are no well-defined strategies that can readily identify PO glycosites, in a complex mixture. We demonstrate a simple, rapid but comprehensive sample preparation platform, the GlycoFilter, which collects N-glycans and peptides separately in a single spin filter device. The lectinbased enrichment method of N-glyco FASP uses a filtration principle to identify lectin-specific glycosites [12], this platform enables efficient downstream characterization of the N-glycans, N-glycosites, and the remaining proteome of a simple or complex biological sample. The GlycoFilter has the additional nonbiased capability to identify PO N-glycosites using a standard LC-MS/MS approach
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