(1) Proteins were extracted from whole body of mixed stages of C.elegans and tissues of male mouse (C57BL/6J) with a denaturing buffer solution (7 M guanidine-HCl). The proteins were S-reduced, alkylated, dialyzed, and then digested with trypsin.

(2) From the digest, glycopeptide subsets were captured using affinity chromatography on a series of immobilized lectin columns or a hydrophilic column (Amide80).

(3) The glycopeptides were treated with peptide-N-glycanase (PNGase) F in a solvent using stable isotope-lebeled water, H₂¹⁸O, to remove Asn-linked glycans and to label the glycosylated Asn residue as Asp incorporating ¹⁸O.

(4) The labeled peptides were separated on a small reverse phase (C18) column and then introduced directly into MS via electrospray ionization interface to acquire their MS/MS spectra.

(5) Based on the spectra data, peptide sequences and the position of glycosylated Asn were determined by database search technology. Please see papers about the method in detail: Kaji H. et al (2003) Nature Biotechnology or (2006) Nature Protocols.