Single-Step Enrichment of N-Glycopeptides and Phosphopeptides with Novel Multifunctional Ti-Immobilized Dendritic Polyglycerol Coated Chitosan Nanomaterials.

Protein glycosylation and phosphorylation, two of the most important post-translational modifications (PTMs) in the proteome, play a vital role in regulating a number of complex biological processes and involvement in a variety of diseases. Comprehensive characterization of the phosphoproteome and glycoproteome requires highly specific and sensitive enrichment methods of purification of phosphopeptides and glycopeptides because many glycoproteins and phosphoproteins naturally occur at low abundances and substoichiometry. Here, we reported a facile route to fabricate a novel multifunctional Ti-mmobilized dentritic polyglycerol CS@PGMA@IDA (CS, chitosan; PGMA, poly(glycidyl methacrylate); IDA, iminodiacetic acid) nanomaterials. The polymer surface endows the nanomaterials with biocompatibility, excellent hydrophilic property, and a large amount of Ti which have the property of immobilized metal ion affinity chromatography (IMAC)- and hydrophilic interaction liquid chromatography (HILIC)-based functional materials. The CS@PGMA@IDA-Ti nanomaterials demonstrate an outstanding ability for N-glycopeptides and phosphopeptides enrichment simultaneously, evaluated by the extremely high binding capacity (150 mg g), sensitivity (above 0.1 fmol), and high enrichment recovery (above 75.4%). Its outstanding specificity and efficiency for purification of phosphopeptides is reflected in quantities as low as 1:5000 molar ratios of phosphopeptides which can be detected. Furthermore, we used CS@PGMA@IDA-Ti to enrich for N-glycopeptides and phosphopeptides followed by PNGase F treatment, fractionated and separated N-glycopeptides and phosphopeptides with different eluents, and then analyzed by MS, a total of 423 (84.4 ID/μg, 3.525 ID/min) N-glycopeptides in 235 different glycoproteins and 422 (84.4 ID/μg, 3.517 ID/min) phosphopeptides in 256 different phosphoproteins which were finally identified in two independent LC-MS/MS runs (with a total time of 120 min) from 50 μg of mouse liver. The results demonstrated that the method based on CS@PGMA@IDA-Ti to single-step enrichment of N-glycopeptides and phosphopeptides is simple, efficient, specific, and compatible to MS. It can be expected that CS@PGMA@IDA-Ti would hold great applicability of modification-based proteomics to the precious and low amounts of clinical samples.