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 Ti4+-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 4+ which have the property of immobilized metal ion affinity chromatography (IMAC)- and hydrophilic interaction liquid chromatography (HILIC)-based functional materials. The CS@PGMA@IDA-Ti4+ nanomaterials demonstrate an outstanding ability for N-glycopeptides and phosphopeptides enrichment simultaneously, evaluated by the extremely high binding capacity (150 mg g–1), 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-Ti4+ 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-Ti4+ 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-Ti4+ would hold great applicability of modification-based proteomics to the precious and low amounts of clinical samples.

CS@PGMA@IDA nanomaterials were facilely synthesized, the zwitterion polymer surface PGMA@IDA endows the nanomaterial with biocompatibility, excellent hydrophilic properties and a large amount of functional groups on the polymer chains that can selectively bind to glycopeptides based on hydrophilic interaction.

In this study we describe a method for highly specific enrichment of glycopeptides with boronic acid-functionalized chitosan polymeric nanospheres and matrix assisted laser desorption-ionization mass spectrometry (MALDI-MS). This is the first time chitosan has been used to create nanosphere support material for selective enrichment of glycopeptides by modification with glycidyl methacrylate (GMA) and derivatization with 3-aminophenylboronic acid (APB). Due to their multifunctional chemical moieties, these 20–100 nm chitosan–GMA–APB nanospheres have unique properties, such as good dispersibility, good biocompatibility and chemical stability, as well as augmented specificity with glycopeptides. Enrichment conditions were optimized by using trypsin digested glycoprotein horseradish peroxidase. The high specificity of chitosan–GMA–APB nanospheres was demonstrated by effectively enriching glycopeptides from a digest mixture of horseradish peroxidase and nonglycoproteins (bovine serum albumin (BSA)).Highlights► This report describes the highly specific capture of glycopeptides employing chitosan–GMA–APB nanospheres. ► These nanospheres have a diameter of 20–100 nm and were synthesized for this work. ► MALDI-TOF-MS combined with use of these nanospheres is a simple and rapid approach for enrichment of glycopeptides. ► The binding affinity of boronic acid and glycopeptides is pH and solvent dependent.

In this study, we describe characterization of the human plasma proteome based on analysis with multifunctional chitosan-GMA-IDA-Cu(II) nanospheres. Chitosan-GMA-IDA-Cu(II) nanospheres with diameters of 20 to 100 nm have unique properties due to multifunctional chemical moieties, high surface area, high capacity, good dispersibility in buffer solution as well as good biocompatibility and chemical stability which improves their specific interaction with peptides and proteins of the human plasma using different binding buffers. Combining these chitosan-GMA-IDA-Cu(II) nanospheres with MS spectrometry results in a novel strategy which should make it possible to characterize the plasma proteome in a single test. Peptides and proteins adsorbed on the nanosphere can be directly detected by MALDI-TOF-MS. The eluted lower molecular weight peptides and proteins are identified by nano-LC-ESI-MS/MS. A total of 842 unique LMW peptides and 1,682 human unredundant proteins IDs were identified in two different binding buffers, which included relatively low-level proteins (e.g., pg/mL of IL3 Interleukin-3) co-distributed with high-abundance proteins (e.g., 35–55 mg/mL level serum albumin). As such, this nanosphere technique selectively enabled the identification of proteins over a dynamic range of greater than 8 orders of magnitude. Considering this capacity for selective enrichment of peptides and proteins in human plasma, and the large number of LMW peptides and proteins which can be identified, this method promises to accelerate discovery of biomarkers for clinical application.

CS@PGMA@IDA nanomaterials were facilely synthesized, the zwitterion polymer surface PGMA@IDA endows the nanomaterial with biocompatibility, excellent hydrophilic properties and a large amount of functional groups on the polymer chains that can selectively bind to glycopeptides based on hydrophilic interaction.