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Interlaboratory study on differential analysis of protein glycosylation by mass spectrometry: the ABRF glycoprotein research multi-institutional study 2012.

ABSTRACT: One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary in order for one to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. The accurate comparison of glycoforms and quantification of glycosites are essential steps in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information. As part of the 2012 ABRF Glycoprotein Research Group study, we explored the use of mass spectrometry and ancillary methodologies to characterize the glycoforms of two sources of human prostate specific antigen (PSA). PSA is used as a tumor marker for prostate cancer, with increasing blood levels used to distinguish between normal and cancer states. The glycans on PSA are believed to be biantennary N-linked, and it has been observed that prostate cancer tissues and cell lines contain more antennae than their benign counterparts. Thus, the ability to quantify differences in glycosylation associated with cancer has the potential to positively impact the use of PSA as a biomarker. We studied standard peptide-based proteomics/glycomics methodologies, including LC-MS/MS for peptide/glycopeptide sequencing and label-free approaches for differential quantification. We performed an interlaboratory study to determine the ability of different laboratories to correctly characterize the differences between glycoforms from two different sources using mass spectrometry methods. We used clustering analysis and ancillary statistical data treatment on the data sets submitted by participating laboratories to obtain a consensus of the glycoforms and abundances. The results demonstrate the relative strengths and weaknesses of top-down glycoproteomics, bottom-up glycoproteomics, and glycomics methods.

SUBMITTER: Leymarie N

PROVIDER: S-EPMC3790302 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Interlaboratory study on differential analysis of protein glycosylation by mass spectrometry: the ABRF glycoprotein research multi-institutional study 2012.

Leymarie Nancy N Griffin Paula J PJ Jonscher Karen K Kolarich Daniel D Orlando Ron R McComb Mark M Zaia Joseph J Aguilan Jennifer J Alley William R WR Altmann Friederich F Ball Lauren E LE Basumallick Lipika L Bazemore-Walker Carthene R CR Behnken Henning H Blank Michael A MA Brown Kristy J KJ Bunz Svenja-Catharina SC Cairo Christopher W CW Cipollo John F JF Daneshfar Rambod R Desaire Heather H Drake Richard R RR Go Eden P EP Goldman Radoslav R Gruber Clemens C Halim Adnan A Hathout Yetrib Y Hensbergen Paul J PJ Horn David M DM Hurum Deanna D Jabs Wolfgang W Larson Göran G Ly Mellisa M Mann Benjamin F BF Marx Kristina K Mechref Yehia Y Meyer Bernd B Möginger Uwe U Neusüβ Christian C Nilsson Jonas J Novotny Milos V MV Nyalwidhe Julius O JO Packer Nicolle H NH Pompach Petr P Reiz Bela B Resemann Anja A Rohrer Jeffrey S JS Ruthenbeck Alexandra A Sanda Miloslav M Schulz Jan Mirco JM Schweiger-Hufnagel Ulrike U Sihlbom Carina C Song Ehwang E Staples Gregory O GO Suckau Detlev D Tang Haixu H Thaysen-Andersen Morten M Viner Rosa I RI An Yanming Y Valmu Leena L Wada Yoshinao Y Watson Megan M Windwarder Markus M Whittal Randy R Wuhrer Manfred M Zhu Yiying Y Zou Chunxia C

Molecular & cellular proteomics : MCP 20130613 10

One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary in order for one to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. The accurate comparison of glycoforms and quantification of glycosites are essential steps in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynam ...[more]

PMID: 23764502

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Project description:Questions concerning longitudinal data quality and reproducibility of proteomic laboratories spurred the Protein Research Group of the Association of Biomolecular Resource Facilities (ABRF-PRG) to design a study to systematically assess the reproducibility of proteomic laboratories over an extended period of time. Developed as an open study, initially 64 participants were recruited from the broader mass spectrometry community to analyze provided aliquots of a six bovine protein tryptic digest mixture every month for a period of nine months. Data were uploaded to a central repository, and the operators answered an accompanying survey. Ultimately, 45 laboratories submitted a minimum of eight LC-MSMS raw data files collected in data-dependent acquisition (DDA) mode. No standard operating procedures were enforced; rather the participants were encouraged to analyze the samples according to usual practices in the laboratory. Unlike previous studies, this investigation was not designed to compare laboratories or instrument configuration, but rather to assess the temporal intralaboratory reproducibility. The outcome of the study was reassuring with 80% of the participating laboratories performing analyses at a medium to high level of reproducibility and quality over the 9-month period. For the groups that had one or more outlying experiments, the major contributing factor that correlated to the survey data was the performance of preventative maintenance prior to the LC-MSMS analyses. Thus, the Protein Research Group of the Association of Biomolecular Resource Facilities recommends that laboratories closely scrutinize the quality control data following such events. Additionally, improved quality control recording is imperative. This longitudinal study provides evidence that mass spectrometry-based proteomics is reproducible. When quality control measures are strictly adhered to, such reproducibility is comparable among many disparate groups. Data from the study are available via ProteomeXchange under the accession code PXD002114.

Dataset Information

Interlaboratory study on differential analysis of protein glycosylation by mass spectrometry: the ABRF glycoprotein research multi-institutional study 2012.

Publications

Interlaboratory study on differential analysis of protein glycosylation by mass spectrometry: the ABRF glycoprotein research multi-institutional study 2012.

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