Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry.
Quantitative proteome profiling using stable isotope protein tagging and automated tandem mass spectrometry (MS/MS) is an emerging technology with great potential for the functional analysis of biological systems and for the detection of clinical diagnostic or prognostic marker proteins. Owing to the enormous complexity of proteomes, their comprehensive analysis is an as-yet-unresolved technical challenge. However, biologically or clinically important information can be obtained if specific, information-rich protein classes, or sub-proteomes, are isolated and analyzed. Glycosylation is the most common post-translational modification. Here we describe a method for the selective isolation, identification and quantification of peptides that contain N-linked carbohydrates. It is based on the conjugation of glycoproteins to a solid support using hydrazide chemistry, stable isotope labeling of glycopeptides and the specific release of formerly N-linked glycosylated peptides via peptide- N-glycosidase F (PNGase F). The recovered peptides are then identified and quantified by MS/MS. We applied the approach to the analysis of plasma membrane proteins and proteins contained in human blood serum.
Zhang
Hui
H
Institute for Systems Biology, 1441 N 34th Street, Seattle, Washington 98103-8904, USA.
Li
Xiao-Jun
XJ
Martin
Daniel B
DB
Aebersold
Ruedi
R
D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D012117:Resins, Synthetic; D000514:alpha 1-Antichymotrypsin / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D000515:alpha 1-Antitrypsin / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D060749:alpha-2-HS-Glycoprotein
C556354:AHSG protein, human; D001798:Blood Proteins; D006023:Glycoproteins; D010455:Peptides; D011134:Polysaccharides; D012117:Resins, Synthetic; D000514:alpha 1-Antichymotrypsin; D000515:alpha 1-Antitrypsin; D060749:alpha-2-HS-Glycoprotein