Comparative Glycopeptide Mapping Study

2012 Comparative Glycopeptide Mapping Study

Invitation

Laboratories engaged in mass spectrometry-based proteomics, glycoproteomics, or glycomics are invited to participate in a glycoprotein analysis study in 2012.  This study was initiated by the Glycoprotein Research Group of the Association for Biomolecular Resource Facilities (ABRF).  Those indicating interest will be sent two sources of prostate specific antigen (PSA) for comparative glycopeptide N-glycosylation analysis.  Samples will be sent in early March, 2012.  The deadline for submission of results will be July 31, 2012.  The results will be communicated in a publication that will be written by the study organizers.  All those submitting data for the study will be included as co-authors for this publication.  The study results will be presented at the ABRF annual conference in 2013 and at other scientific meetings.    

To participate please email your interest to Joe Zaia (jzaia@bu.edu) and include your shipping address.

Study goals

The goal of this study is to determine the ability of the glycoproteomics community to compare N-glycosylation between two different sources of prostate specific antigen (PSA).  The PSA sources have been selected by the study organizers and the differences in glycosylation are known.  

Background

Accurate mapping of glycoprotein glycosylation is essential for basic glycosciences, biomarker discovery, and recombinant glycoprotein therapeutic characterization.  For these purposes, it is necessary to identify and determine the abundances of glycopeptides derived from the target protein.  This glycopeptide mapping insures that data are produced from the target proteins rather than from contaminants.   N-Linked glycopeptides are present typically as a series of glycoforms, all containing the chitobiose core.  Because proteolytic digestion often results in glycopeptides containing incomplete cleavage sites, the abundances of glycopeptide glycoforms must be reconstructed from those of several different ions.  For these reasons, accurate glycopeptide mapping represents an analytical challenge.  

Study rationale

This is a glycopeptide mapping study.  For the purpose of accurate determination of glycoprotein glycosylation, it is necessary to determine glycosylation on specific peptides.  Most glycoprotein samples are not pure.  Contamination with other proteins, even at a level of a few percent, may give rise to false positive identifications.  Thus, for the purpose of identifying the peptide and glycan parts, it is important to determine the masses of intact glycopeptides.  Investigators may also wish to analyze released glycans.  The study will compare results obtained using glycopeptides with those from released glycans.

Analysis recommendations

Store the PSA at 4°C.  Freezing and drying of the sample is not recommended.  Freezing of proteolytically digested samples is not recommended.

Data reporting

Participants should report their results using an Excel template that will be provided by the organizing committee.   To use this template, the data should be converted into glycan compositions (Hex, HexNAc, dHex, NeuAc, NeuGc, Sulfate, Phosphate) that modify the PSA N-glycosylation site.

Study sponsors

PSA was donated generously by Lee Biosolutions.  Support for the costs of the study was provided by the Association for Biomolecular Resource Facilities, Thermo-Fisher Scientific, and Bruker Daltonics, Inc.

We look forward to your participation.  Sincerely,

Study organizers

Nancy Leymarie                     (Center for Biomedical Mass Spectrometry, Boston University)

Joseph Zaia                 (Center for Biomedical Mass Spectrometry, Boston University)

Ron Orlando              (Complex Carbohydrate Research Center, University of Georgia)

Daniel Kolarich                      (Max Plank Institute of Colloids and Interfaces)

Karen Jonscher                       (University of Colorado, Denver)