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)
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