O-glycan analysis by multi-tandem MS

 A cell lysate (~500 μg) is precipitated with cold acetone (3–5 times volume of lysate).

 The precipitate is collected by centrifugation and the residue is dried with a Speed-Vac

 The dried precipitates are disolved in 7 M guanidine hydrochloride, 0.5 M Tris-HCl (pH 8.6), 10 mM EDTA-Na and are reduced with 10 mM dithiothreitol followed by alkylation with 20 mM iodoacetamide.

 Sample is dialyzed against 10 mM NH₄HCO₃ using a Mini Dialysis Kit with a molecular mass cut-off of 8 kDa.

 The dialyzed sample is digested with 25 μg trypsin at 37ºC overnight, followed by heat inactivation of the trypsin.

 5–10 mU PNGaseF are added to the solutions, followed by incubation at 37°C overnight to remove N-glycans

 The reaction solution is passed through a polymeric reversed-phase column (e.g. Oasis HLB), and O-glycans are directly subjected to reductive β-elimination on the column.

 100 μL 500 mM NaBH ₄ in 50 mM NaOH are applied to the column and incubated at 50°C overnight to release O-glycans.

 The released O-glycans are passed through Oasis HLB column and neutralized by adding 10 μL of 50% aqueous acetic acid.

 The released and reduced O-glycans are desalted by a cation-exchange column (e.g.Oasis MCX) or resin (e.g. AG 50W-X8 Resin).

 The desalted are eluted with 750 μL of distilled water and dried with a Speed-Vac.

 The remaining borate is removed by the addition of 50–100 μL of 1% acetic acid in methanol and dried under vacuum several times.

 The NaOH small beads (approximately 50 mg) are mixed with 250 μL of DMSO (∞Pure; Wako Pure Chemical Industries, Ltd.).

 Approximately 50 μL of the slurry is added to the released O-glycans (alditols) in a glass tube followed by 50 μL iodomethane (Wako Pure Chemical Industries, Ltd.) and the mixture is agitated at room temperature for 30 min.

 The reaction is terminated by addition of 1 mL of 1 % aqueous HCl (or 1 mL of 10% aqueous acetic acid) on the ice.

 The diluted reaction solution is applied to a reversed-phased column (e.g. Sep-Pak C18 or Oasis HLB) and washed with 2 mL of distilled water.

 The permethylated alditols are eluted with 500–600 μL of 95% aqueous acetonitrile and dried with a Speed-Vac.

 The dried permethylated glycans are resuspended in 25–50 mL acetonitrile.

 0.5 μL of matrix solution (10 mg of 2,5-DHB dissolved in 1 mL of 50% ethanol*) and 0.5–1 μL of the diluted analyte solution are spotted on the MALDI plate (stainless steel plate) and mixed on the plate.

*matrix solution for the stainless steel target with hydrophilic islands (focus spots) on a hydrophobic surface (e.g. AnchorChip plate or μFocus plate): 1 mg of 2,5-DHB dissolved in 1 mL of 50% ethanol

 The dried matrix-analyte mixture is recrystallized with 1 μL of ethanol.

 MSⁿ spectra are carried out in reflectron positive ion mode and obtained from Na adduct ions.