Screening for <b><i>N</i></b>‐glycosylated proteins by liquid chromatography mass spectrometry

Bunkenborg, Jakob; Pilch, Bartosz; Podtelejnikov, Alexandre V.; Wiśniewski, Jacek R.

doi:10.1002/pmic.200300556

Cited by 181 publications

(177 citation statements)

References 31 publications

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“…Using the urological 2-DE databases, pathologic subtypes of solid bladder tumor tissue specimens could be distinguished on the basis of protein fingerprints. 12,20,21 Although a number of dysregulated proteins have been identified in a variety of tissue-based studies, it is disappointing that no reliable markers have been identified for transitional cell carcinoma, the most common type of bladder cancer. Through the proteomic study of a set of only six urine samples, Kageyama et al 22 were able to identify a potential tumor marker, calreticulin, which is found in the urine of patients with bladder carcinoma.…”

Section: Discussionmentioning

confidence: 99%

Bladder Cancer Associated Glycoprotein Signatures Revealed by Urinary Proteomic Profiling

et al. 2007

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Current methods in the noninvasive detection and surveillance of bladder cancer via urine analysis include voided urine cytology (VUC) and some diagnostic urinary protein biomarkers; however, due to the poor sensitivity of VUC and high false-positive rates of currently available protein assays, detection of bladder cancer via urinalysis remains a challenge. In the study presented here, a rapid, high-sensitivity technique was developed to profile the N-linked glycoprotein component in naturally micturated human urine specimens. Concanavalin A (Con A) affinity chromatography coupled to nanoflow liquid chromatography was utilized to separate the complex peptide mixture prior to a linear ion trap MS analysis. Of 186 proteins identified with high confidence by multiple analyses, 40% were secreted proteins, 18% membrane proteins, and 14% extracellular proteins. In this study, the presence of several proteins appeared to be associated with the presence of bladder cancer, including R-1B-glycoprotein that was detected in all tumor-bearing patient samples but in none of the samples obtained from nontumor-bearing individuals. The combination of Con A affinity chromatography and nano-LC/MS/MS provides an initial investigation of N-glycoproteins in complex biological samples and facilitates the identification of potential biomarkers of bladder cancer in noninvasively obtained human urine.

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Section: Discussionmentioning

confidence: 99%

Bladder Cancer Associated Glycoprotein Signatures Revealed by Urinary Proteomic Profiling

et al. 2007

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“…The use of LAC for isolating both intact glycoproteins and glycopeptides (after proteolytic digestion) can further enhance the identification of glycoproteins. Using these affinity capturing steps and LC-MS/MS, a total of 86 Nglycosylation sites in 77 proteins in human serum were identified [70]. SEC was also found useful for significant enrichment of N-linked glycopeptides relative to nonglycosylated peptides because the N-linked glycans expressed on tryptic glycopeptides contribute substantially to their mass [71].…”

Section: Glycoproteins and Lipoproteinsmentioning

confidence: 99%

“…Lectin affinity chromatography (LAC) is often used for enrichment of either glycoproteins or glycopeptides prior to MS analysis [67][68][69][70]. The use of multilectin affinity column maximized the capturing of glycoproteins and therefore yielded a significant list of 150 glycoproteins in serum/plasma.…”

Section: Glycoproteins and Lipoproteinsmentioning

confidence: 99%

Human body fluid proteome analysis

2006

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The focus of this article is to review the recent advances in proteome analysis of human body fluids, including plasma/serum, urine, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, synovial fluid, nipple aspirate fluid, tear fluid, and amniotic fluid, as well as its applications to human disease biomarker discovery. We aim to summarize the proteomics technologies currently used for global identification and quantification of body fluid proteins, and elaborate the putative biomarkers discovered for a variety of human diseases through human body fluid proteome (HBFP) analysis. Some critical concerns and perspectives in this emerging field are also discussed. With the advances made in proteomics technologies, the impact of HBFP analysis in the search for clinically relevant disease biomarkers would be realized in the future.

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“…94 proteins were identified in previous urine proteome studies (1)(2)(3)(4)(5). 43 proteins were also identified in serum Nglycoproteome (11)(12)(13). 150 were annotated as glycoproteins or subunits of glycoproteins in Swiss-Prot, and 43 were annotated as potential glycoproteins predicted by NetNGlyc 1.0.…”

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confidence: 99%

Concanavalin A-captured Glycoproteins in Healthy Human Urine

Wang

Sun

et al. 2006

Molecular & Cellular Proteomics

115

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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...

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Screening for N‐glycosylated proteins by liquid chromatography mass spectrometry

Cited by 181 publications

References 31 publications

Bladder Cancer Associated Glycoprotein Signatures Revealed by Urinary Proteomic Profiling

Bladder Cancer Associated Glycoprotein Signatures Revealed by Urinary Proteomic Profiling

Human body fluid proteome analysis

Concanavalin A-captured Glycoproteins in Healthy Human Urine

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