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, 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.

Fig. 1.

The experimental workflow of the GlycoFilter platform. The entire platform comprises three consecutive steps: preprocessing, de-N-glycosylation, and proteolysis. Each step involves separation by high-speed centrifugation and filtration. During the initial preprocessing step, the proteins are reduced and alkylated, and excess reagents are removed by the first round of filtration. The de-N-glycosylation is catalyzed by PNGase F in an H₂¹⁸O environment to label the de-N-glycosylation site in the protein backbone with one atom of ¹⁸O. The released N-glycans are collected by the second round of filtration. The proteolysis is catalyzed by trypsin, and the tryptic peptides are collected by the third round of filtration. Both enzymatic reactions can be accelerated by the assistance of a domestic microwave, resulting in a dramatic decrease of the overall processing time.

Fig. 2.

The MALDI-MS spectra of permethylated N-glycans released from bovine ribonuclease B (A), bovine fetuin (B), and urine sample U1 (C), collected by the GlycoFilter platform. The Neu5Gc peaks in (B) are annotated with an*. The compositions of the two largest glycans (m/z 4324.9 and 4412.9) in (B) were further confirmed by high resolution ESI-MS and MS/MS fragmentation spectra (Supplemental Fig. S3). All peaks in U1 (C) were assigned a putative topology based on the N-glycosylation biosynthetic pathway and their m/z values. All peaks were single sodium adduct and the monoisotopic peak was annotated.

Fig. 3.

The comparisons of three different urinary proteomes (U1, U2, and U3) with and without PNGase F (+PNGase F versus Control, supplemental Fig. S6) to determine the impact of upfront de-N-glycosylation on overall proteomic performance. A, A Venn-Diagram comparison between +PNGase F and Control at the peptide and the protein levels. The numbers in parentheses indicate the deglycopeptides or glycoproteins identified. In B and C the x axis indicates the log₂ value of the ratio of unique peptide counts for either glycoproteins (B) or other proteins (C) identified in common between +PNGase F and Control. The y axis designated the number of proteins within that specific ratio.

Fig. 4.

Two distinct MS/MS spectra of the peptide sequence “DLDMFINASK” of the glycoprotein attractin (O75882, ATRN_HUMAN) confirmed a PO glycosite at asparagine-1198. A, The position of the nonsugar-modified asparagine within the sequence was identified by the y4/b7 ions (the precursor ion: m/z 577.2818 ²⁺). B, The position of de-N-glycosylated asparagine (¹⁸O-incorporated aspartic acid) was identified by the 3 Da mass shift of a series of b- and y- ions (red highlight) (the precursor ion: m/z 578.7756 ²⁺)The nomenclature of peptide fragment ions was based on previous report (45).