O-Glycan Profiling of Secreted Mucin Protein.

All reagents were purchased from Sigma unless otherwise mentioned. Purified mucin proteins (600 μg, each) was denatured by heating at 100 °C for 5 min. The denatured proteins were subsequently treated with 19 mg of sodium borohydride (NaBH₄) in 500 μL of 50 mM sodium hydroxide (NaOH) solution at 45 °C for 18 h.⁵⁴ The samples were cooled, neutralized with 10% acetic acid, passed through a Dowex H+ resin column, and lyophilized with borates removed under the stream of nitrogen. The glycans were permethylated for structural characterization by mass spectrometry using previously reported methods.⁵⁵ Briefly, the dried eluate was dissolved with dimethyl sulfoxide (DMSO) and methylated by using methyl iodide and NaOH–DMSO base (prepared by mixing DMSO and 50% w/w NaOH solution). The reaction was quenched with water and the reaction mixture was extracted with methylene chloride and dried. The permethylated glycans were dissolved in methanol and crystallized with α-dihydroxybenzoic acid (DHBA, 20 mg/mL in 50% v/v methanol/water) matrix. Analysis of glycans present in the samples was performed in the positive ion mode by MALDI-TOF/TOF-MS using an AB SCIEX TOF/TOF 5800 (Applied Biosystem, MDS Analytical Technologies) mass spectrometer. Permethylated glycans from the samples were infused on an Orbitrap Fusion Tribrid mass spectrometer through an electrospray (ESI) probe with HCD and CID fragmentation option for further structural confirmation. The MS1 and MS2 spectra of the glycans were acquired at high resolution by a simple precursor scan, and respective ions were selected manually for further MS/MS scanning. Assignment of glycan structures were done manually and by using Glycoworkbench software, based on the fragmentation patterns and common biosynthetic pathways.