Hydrazide resin

Viable cells were incubated with 1 mM sodium periodate for 10 min to oxidize glycoproteins [66 (link)]. Oxidized glycoproteins were conjugated to hydrazide resin (Bio-Rad, Hercules, CA) at 4 °C overnight [67 (link)]. After washing sequentially with: 2 M NaCl, 2 % SDS, 200 mM propanolamine (0.1 M NaAcetate, pH 5.5), 40 % ethanol and 80 % ethanol; bound proteins were reduced with dithiothreitol, and alkylated with ICAT™ reagent (Life Technologies/Thermo Fisher Scientific, Applied Biosystems, Framingham, MA). Alkylated proteins were digested with trypsin and cysteine-containing peptides were captured using an avidin column (Life Technologies/Thermo Fisher Scientific). In addition to the cysteine-containing peptide fraction, peptides bound to the resin were also collected and analyzed. Release of peptides was achieved through PNGase-F digestion (New England BioLabs, Ipswich, MA.). While we found some overlap between the proteins identified in the two fractions, analysis of both the cysteine -containing fraction and the resin-bound fraction resulted in complementary coverage of the cell surface protein population.

Hydrazide resin (Bio-Rad, Hercules, CA), sodium periodate (Bio-Rad, Hercules, CA), tris (2-carboxyethyl) phosphine (TCEP) (Pierce, Rockford, IL), PNGase F (New England Biolabs, Ipswich, MA), sequencing grade trypsin (Promega, Madison, WI), C18 columns (Waters, Sep-Pak Vac) were used in our experiment. Additional lab supply and chemicals were purchased from Sigma-Aldrich. iTRAQ reagent and mass calibration standards were from AB SCIEX (Foster City, CA); BCA assay kit was from Pierce (Rockford, IL).

Based on specific capture of glycoproteins by hydrazide resin, the N-glycosylation sites were analyzed and identified using the LC–MS/MS (Thermo Scientific, MA, USA) (Zhang et al. 2003 (link)) (Fig. 1). 1 mg EcXly were dissolved in coupling buffer (100 mM NaAc, 150 mM NaCl), and 15 mM NaIO₄ was added to oxidize the proteins for 1 h. NaIO₄ was then removed using the ultrafiltration tube, and the proteins were coupled with hydrazide resin (Bio-Rad, USA) at room temperature for 10–24 h. To remove the nonglycoproteins, the sample was washed six times using an equal volume of buffer. The proteins were reduced by adding 20 mM dithiothreitol at 60 °C for 1 h and subsequently alkylated by the addition of 20 mM iodoacetamide in the dark for 40 min. The trypsin was used to hydrolyze proteins into peptides, and the PNGase F was used to release the enriched glycopeptides from hydrazide resin. Finally, the released peptides were desalinated and resuspended in an appropriate amount of 0.1% formic acid for further LC–MS/MS analysis. The peptides of MiXly and the mutants of EcXly were obtained by in-gel digestion and used for LC–MS/MS analysis (De Godoy et al 2006 (link)). The acquired MS data were analyzed using Proteome Discoverer 2.2.1 software as previously described (Yuan et al. 2022 (link)).Fig. 1

The schematic workflow of glycosylation analysis using LC–MS/MS

The N-glycopeptides of salivary proteins were enriched by the hydrazide method according to previously described protocols [51 ,52 (link)]. Briefly, half of the peptides (~0.5 mg) from each group were oxidized by 10 mM sodium periodate at room temperature for 1 h in the dark. The oxidized peptide samples were diluted 16-fold with 0.1% TFA and purified by C18 column. The peptides were eluted directly into hydrazide resin (Bio-Rad, Hercules, CA) and incubated overnight at room temperature with shaking.
The resin was washed three times each with 80% ACN, 1.5 M NaCl and D.I. water. The formerly N-linked glycopeptides were released from resin via 2 μL PNGase F (New England Biolabs, Beverly, MA) in 100 μl of 25 mM ammonium bicarbonate at 37°C overnight with shaking. The supernatant and wash solutions were combined and dried via SpeedVac.

Hydrazide chemistry approach was used to enrich the N-glycopeptides as described in a previous study44 (link), with modifications. The tryptic peptides of all samples were dissolved in coupling buffer at pH 5.5 (100 mM NaOAc and 150 mM NaCl) and then an oxidizing agent (sodium periodate (15 mM), Sigma-Aldrich, St. Louis, MO) was added to the samples. The reaction mixture was incubated for 30 min in the dark, and the excess oxidant was quenched with sodium sulfite (20 mM) for 10 min. The hydrazide resin (Bio-Rad, Hercules, CA) was added (20 mg/mL) to the peptides solution and subjected to the coupling reaction overnight at 37 °C. Two sequential washings of the hydrazide resin was performed with water, 1.5 M NaCl, MeOH, and 80% ACN. PNGase F obtained from New England Biolabs was used (1 μL/2–6 mg of crude protein) to release the resin bound glycans from N-glycopeptides. The PNGase F reaction was performed by overnight incubation at 37 °C in 100 mM NH4HCO3 solution. Finally, the N-glycopeptides were obtained by collecting the supernatant (A). In addition, the resin was washed with an 80% ACN and supernatant (B) was collected, and then supernatants A and B were mixed. The samples were desalted using a C18 column prior to the LC-MS/MS analysis.