title: An improved in-gel digestion method for efficient identification of protein and glycosylation analysis of glycoproteins using guanidine hydrochloride.
abstract: In-gel digestion followed by LC/MS/MS is widely used for the identification of trace amounts of proteins and for the site-specific glycosylation analysis of glycoproteins in cells and tissues. A major limitation of this technique is the difficulty in acquiring reliable mass spectra for peptides present in minute quantities and glycopeptides with high heterogeneity and poor hydrophobicity. It is considered that the SDS used in electrophoresis can interact with proteins noncovalently and impede the ionization of peptides/glycopeptides. In this study, we report an improved in-gel digestion method to acquire reliable mass spectra of a trace amount of peptides/glycopeptides. A key innovation of our improved method is the use of guanidine hydrochloride, which forms complexes with the residual SDS molecules in the sample. The precipitation and removal of SDS by addition of the guanidine hydrochloride was successful in improving the S/N of peptides/glycopeptides in mass spectra and acquiring a more comprehensive MS/MS data set for the various glycoforms of each glycopeptide.
authors.affiliation: Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Takakura
authors.forename: Noritaka
authors.initials: N
authors.lastname: Hashii
authors.forename: Nana
authors.initials: N
authors.lastname: Kawasaki
mesh_terms: D000595:Amino Acid Sequence; D011232:Chemical Precipitation; D002853:Chromatography, Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006020:Glycopeptides / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D019791:Guanidine / Q000737:chemistry*; D008969:Molecular Sequence Data; D040901:Proteomics / Q000379:methods; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry* / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D006020:Glycopeptides; D006023:Glycoproteins; D012967:Sodium Dodecyl Sulfate; D019791:Guanidine
keywords: Glycoproteomics; Glycosylation analysis; Guanidine hydrochloride; In-gel digestion; LC/MS