Concanavalin A-captured Glycoproteins in Healthy Human Urine

Abstract

Both the urinary proteome and its glycoproteome can reflect human health status, and more directly, functions of kidney and urinary tracts. Because the high abundance protein albumin is not N-glycosylated, the urine N-glycoprotein enrichment procedure could deplete it, and urine proteome could thus provide a more detailed protein profile in addition to glycosylation information especially when albuminuria occurs in some kidney diseases. In terms of describing the details of urinary proteins, the urine glycoproteome is even a better choice than the proteome itself. Pooled urine samples from healthy volunteers were collected and acetone-precipitated for proteins. N-Linked glycoproteins enriched with concanavalin A affinity purification were separated and analyzed by SDS-PAGE-reverse phase LC/MS/MS or two-dimensional LC/MS/MS. A total of 225 urinary proteins were identified based on two-hit criteria with reliability over 97% for each peptide. Among these proteins, 94 were identified in previous urine proteome works, 150 were annotated as glycoproteins in Swiss-Prot, and 43 were predicted as glycoproteins by NetNGlyc 1.0. A number of known biomarkers and disease-related glycoproteins were identified. Because changes in protein quantity or the glycosylation status can lead to changes in the concanavalin A-captured glycoprotein profile, specific urine glycoproteome patterns might be observed for specific pathological conditions as multiplex urinary biomarkers. Knowledge of the urine glycoproteome is important in understanding kidney and body function. Molecular & Cellular Proteomics 5:560 -562, 2006.The urinary proteome has received more and more attention in the proteomic field for its simplicity compared with serum as well as its potential in biomarker discovery. Several research teams have worked on profiling the healthy human urine proteome using electrophoresis and/or liquid chromatography followed by mass spectrometry identification (1-5).The fact that some proteins were observed at higher molecular weight than their theoretical ones on SDS-PAGE (5) confirmed that the post-translational modifications including glycosylation exist extensively in urinary proteins. Glycosylation, a common and important protein post-translational modification, is involved in many biological processes such as cell adhesion, signal transduction, immune response, and inflammatory reaction (6). More than half of all proteins are thought to be glycoproteins, and they will undergo changes in quality and quantity along with the changes in different physiological and pathological states. Both the urine proteome and its glycoproteome can reflect human health status, especially functions of kidney and urinary tracts. In some kidney diseases, albuminuria usually occurs. Because high abundance albumin is not N-glycosylated, urine N-glycoprotein enrichment procedure could deplete it, and the urine glycoproteome could provide a more detailed protein profile in addition to the glycosylation information. In terms of describing details of urinary pr...

Applications of urinary proteomics in biomarker discovery

“…Wang et al [6] identified 225 N-glycoproteins in Con-A enriched normal human urinary proteome, whereas Li et al [4] identified 31 phosphoproteins.…”

Discovery of Urinary Biomarkers

“…Large scale proteomics profiling of normal human urine samples has revealed the presence of at least 1000 different protein gene products and many more peptide fragments of larger proteins (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). There is hope, therefore, for discovery of urinary protein excretion profiles that can be used clinically for tasks such as early detection of disease, classification of disease, choice of therapeutic agents, assessment of prognosis, and monitoring of a particular therapeutic regimen.…”

Human Urinary Glycoproteomics; Attachment Site Specific Analysis of N- and O-Linked Glycosylations by CID and ECD

“…Urine is potentially a rich source for N-and Olinked glycoproteins derived from renal-and distal organs and represents an interesting subproteome for structural charac-terization of human glycoproteins. However, glycoproteomic characterization of urine is lacking and only a few proteomic studies aimed at identifying urinary glycoproteins have been reported (17)(18)(19)(20). In these studies, the glycan moieties were either cleaved off or not studied at all.…”