Universal Solid-Phase Reversible Sample-Prep for Concurrent Proteome and N-Glycome Characterization

Abstract

We describe a novel solid-phase reversible sample-prep (SRS) platform that enables rapid sample preparation for concurrent proteome and N-glycome characterization for nearly all protein samples. SRS utilizes a uniquely functionalized, silica-based bead that has strong affinity toward proteins with minimal to no affinity for peptides and other small molecules. By leveraging this inherent size difference between proteins and peptides, SRS permits high-capacity binding of proteins, rapid removal of small molecules (detergents, metabolites, salts, peptides, etc.), extensive manipulation including enzymatic and chemical treatments on bead-bound proteins, and easy recovery of N-glycans and peptides. SRS was evaluated in a wide range of samples including glycoproteins, cell lysate, murine tissues, and human urine. SRS was also coupled to a quantitative strategy to investigate the differences between DU145 prostate cancer cells and its DIAPH3-silenced counterpart. Previous studies suggested that DIAPH3 silencing in DU145 induced transition to an amoeboid phenotype that correlated with tumor progression and metastasis. In this pilot study we identified distinct proteomic and N-glycomic alterations between them. A metastasis-associated tyrosine kinase receptor ephrin-type-A receptor (EPHA2) was highly up-regulated in DIAPH3-silenced cells, indicating a possible connection between EPHA2 and DIAPH3. Moreover, distinct alterations in the N-glycome were identified, suggesting cross-links between DIAPH3 and glycosyltransferase networks.

Keywords: N-glycome; glycoprotein; membrane protein; plasma; proteome; reversible solid phase; sample-prep; urine.

FIG. 1

The SRS workflow separates and captures purified free N-glycans and tryptic peptides for downstream mass spectrometry (MS) analyses. SRS has high binding capacity with all biological samples, including membrane-enriched proteins solubilized in 2% SDS. Non-protein compounds such as metabolites, reagents, salts, and detergents are rapidly removed in minutes. In this workflow, samples undergo sequential enzymatic de-N-glycosylation to release and capture N-glycans, followed by proteolytic digestion to recover peptides. H2O¹⁸ is used to label the original sites of N-glycosylation. The handling times are reported, excluding the time for enzymatic reactions.

FIG. 2

The evaluation of SRS beads on protein binding, buffer washing and enzyme accessibility. (a) Nearly 1 mg of horse heart cytochrome C was bound to 10 mg of dry beads, demonstrating excellent binding of a small and hydrophilic polypeptide. (b) After protein immobilization, efficient and rapid removal of urinary non-protein components was achieved with a rapid two-stage wash. (c) SRS bound bovine fetuin was completely reduced and alkylated, and de-N-glycosylated with PNGase F, achieving results comparable to the classic solutionphase procedure (R/A: reduction and alkylation of disulfide bonds). The intact bovine fetuin was recovered by a mild stripping protocol (Experimental section).

FIG. 3

(a) SRS was employed to quantitatively compare the proteome and N-glycome of DIAPH3-silenced (amoeboid, DU145KD) and non-silenced (control, DU145Ctrl) human prostate cancer cell lines. Released N-glycans were compared by LC-MS utilizing a pair of light and heavy isotopes incorporated tags with a 6 Da mass difference (DRAG)(8). Proteins were quantitatively compared by spectral counting. (b) A representative LC-MS spectrum used to calculate the relative ratio of the same N-glycan species (Hex3HexNAc4) identified in both cell lines. (c) Distinct quantitative ratios were determined among five specific N-glycan subgroups: high-mannose (green), complex-type with unmodified non-reducing end HexNAc (blue), biantennary with terminal sialic acid (light green), triantennary with terminal sialic acid (purple), and tetraantennary with terminal sialic acid (pink). All putative topologies were proposed based on the mammalian N-glycosylation biosynthetic pathway and their m/z values (<10 ppm). Selected compositions were further manually validated by MS/MS fragmentations. H: hexose; N: N-acetylhexosamine; A: N-acetylneuraminic acid (Neu5Ac); F: fucose. (d) Differential expression of selected proteins identified by SRS from DU145Ctrl and DU145KD cell lines. Additional validation of EphA2 expression differences by immunoblotting (e), and immunofluorescence (f). Scale bar, 10μm.