title: Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry.
abstract: The enormous complexity, wide dynamic range of relative protein abundances of interest (over 10 orders of magnitude), and tremendous heterogeneity (due to post-translational modifications, such as glycosylation) of the human blood plasma proteome severely challenge the capabilities of existing analytical methodologies. Here, we describe an approach for broad analysis of human plasma N-glycoproteins using a combination of immunoaffinity subtraction and glycoprotein capture to reduce both the protein concentration range and the overall sample complexity. Six high-abundance plasma proteins were simultaneously removed using a pre-packed, immobilized antibody column. N-linked glycoproteins were then captured from the depleted plasma using hydrazide resin and enzymatically digested, and the bound N-linked glycopeptides were released using peptide-N-glycosidase F (PNGase F). Following strong cation exchange (SCX) fractionation, the deglycosylated peptides were analyzed by reversed-phase capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Using stringent criteria, a total of 2053 different N-glycopeptides were confidently identified, covering 303 nonredundant N-glycoproteins. This enrichment strategy significantly improved detection and enabled identification of a number of low-abundance proteins, exemplified by interleukin-1 receptor antagonist protein (approximately 200 pg/mL), cathepsin L (approximately 1 ng/mL), and transforming growth factor beta 1 (approximately 2 ng/mL). A total of 639 N-glycosylation sites were identified, and the overall high accuracy of these glycosylation site assignments as assessed by accurate mass measurement using high-resolution liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR) is initially demonstrated.
authors.affiliation: Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99354, USA.
authors.forename: Tao
authors.initials: T
authors.lastname: Liu
authors.forename: Wei-Jun
authors.initials: WJ
authors.lastname: Qian
authors.forename: Marina A
authors.initials: MA
authors.lastname: Gritsenko
authors.forename: David G
authors.initials: DG
authors.lastname: Camp
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Monroe
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Moore
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry*; D002412:Cations; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D006020:Glycopeptides / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D006834:Hydrazines / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared; D013347:Subcellular Fractions
chemical_list: D001798:Blood Proteins; D002412:Cations; D006020:Glycopeptides; D006023:Glycoproteins; D006834:Hydrazines; D010455:Peptides; D011506:Proteins; D020543:Proteome; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase