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Interlaboratory Study on Differential Analysis of Protein Glycosylation by Mass Spectrometry: The ABRF Glycoprotein Research Multi-Institutional Study 2012

Journal article
Authors N. Leymarie
P. J. Griffin
K. Jonscher
D. Kolarich
R. Orlando
M. McComb
J. Zaia
J. Aguilan
W. R. Alley
F. Altmann
L. E. Ball
L. Basumallick
C. R. Bazemore-Walker
H. Behnken
M. A. Blank
K. J. Brown
S. C. Bunz
C. W. Cairo
J. F. Cipollo
R. Daneshfar
H. Desaire
R. R. Drake
E. P. Go
R. Goldman
C. Gruber
Adnan Halim
Y. Hathout
P. J. Hensbergen
D. M. Horn
D. Hurum
W. Jabs
Göran Larson
M. Ly
B. F. Mann
K. Marx
Y. Mechref
B. Meyer
U. Moginger
C. Neususs
Jonas Nilsson
M. V. Novotny
J. O. Nyalwidhe
N. H. Packer
P. Pompach
B. Reiz
A. Resemann
J. S. Rohrer
A. Ruthenbeck
M. Sanda
J. M. Schulz
U. Schweiger-Hufnagel
Carina Sihlbom
E. Song
G. O. Staples
D. Suckau
H. X. Tang
M. Thaysen-Andersen
R. I. Viner
Y. M. An
L. Valmuv
Y. Wada
M. Watson
M. Windwarder
R. Whittal
M. Wuhrer
Y. Y. Zhu
C. X. Zou
Published in Molecular & Cellular Proteomics
Volume 12
Issue 10
Pages 2935-2951
ISSN 1535-9476
Publication year 2013
Published at Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine
Core Facilities, Proteomics
Pages 2935-2951
Language en
Subject categories Biochemistry


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. T6G 2G2, Canada. [Cipollo, John F.; An, Yanming] US FDA, Ctr Biol Evaluat & Res, Bethesda, MD 20993 USA. [Desaire, Heather; Go, Eden P.] Univ Kansas, Lawrence, KS 66045 USA. [Goldman, Radoslav; Pompach, Petr; Sanda, Miloslav] Georgetown Univ, Dept Oncol, Washington, DC [Halim, Adnan; Larson, Goran; Nilsson, Jonas] Univ Gothenburg, Sahlgrenska Acad, Dept Clin Chem & [Hensbergen, Paul J.; Wuhrer, Manfred] Leiden Univ, Med Ctr, Biomol Mass Spectrometry Unit, NL- [Jabs, Wolfgang; Marx, Kristina; Resemann, Anja; Schweiger-Hufnagel, Ulrike; Suckau, Detlev] Bruker [Ly, Mellisa; Staples, Gregory O.] Agilent Technol, Agilent Labs, Santa Clara, CA 95051 USA. [Mechref, Yehia; Song, Ehwang] Texas Tech Univ, Dept Chem & Biochem, Lubbock, TX 79409 USA. [Nyalwidhe, Julius O.; Watson, Megan] Eastern Virginia Med Sch, Leroy T Canoles Jr Canc Res Ctr, Dept [Packer, Nicolle H.; Thaysen-Andersen, Morten] Macquarie Univ, Dept Chem & Biomol Sci, Biomol [Sihlbom, Carina] Gothenburg Univ, Prote Core Facil, Gothenburg, Sweden. [Tang, Haixu] Indiana Univ, Sch Informat, Bloomington, IN 47405 USA. [Valmuv, Leena] Finnish Red Cross Blood Serv, Helsinki 00310, Finland. [Wada, Yoshinao] Osaka Med Ctr Maternal & Child Hlth, Res Inst, Izumi Ku, Osaka 5941101, Japan.

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