Quantitative mapping of glycoprotein micro‐heterogeneity and macro‐heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides |
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Authors: | K. Stavenhagen H. Hinneburg M. Thaysen‐Andersen L. Hartmann D. Varón Silva J. Fuchser S. Kaspar E. Rapp P. H. Seeberger D. Kolarich |
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Affiliation: | 1. Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, , 14424 Potsdam, Germany;2. Department of Chemistry and Biomolecular Sciences, Macquarie University, , Sydney, 2109 New South Wales, Australia;3. Bruker Daltonics, , Bremen, Germany;4. Max Planck Institute for Dynamics of Complex Technical Systems, , Magdeburg, Germany;5. Free University Berlin, , Berlin, Germany |
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Abstract: | Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached to particular protein glycosylation sites (micro‐heterogeneity) and evaluate the molar site occupancy (macro‐heterogeneity) in glycoproteomics. However, the accuracy of MS for such quantitative measurements remains to be clarified. As a key step towards this goal, a panel of related tryptic peptides with and without complex, biantennary, disialylated N‐glycans was chemically synthesised by solid‐phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A and endo D/F/H were synthetically produced, carrying aspartic acid and N‐acetylglucosamine‐linked asparagine residues, respectively, at the glycosylation site. The MS ionisation/detection strengths of these pure, well‐defined and quantified compounds were investigated using various MS ionisation techniques and mass analysers (ESI‐IT, ESI‐Q‐TOF, MALDI‐TOF, ESI/MALDI‐FT‐ICR‐MS). Depending on the ion source/mass analyser, glycopeptides carrying complex‐type N‐glycans exhibited clearly lower signal strengths (10–50% of an unglycosylated peptide) when equimolar amounts were analysed. Less ionisation/detection bias was observed when the glycopeptides were analysed by nano‐ESI and medium‐pressure MALDI. The position of the glycosylation site within the tryptic peptides also influenced the signal response, in particular if detected as singly or doubly charged signals. This is the first study to systematically and quantitatively address and determine MS glycopeptide ionisation/detection strengths to evaluate glycoprotein micro‐heterogeneity and macro‐heterogeneity by label‐free approaches. These data form a much needed knowledge base for accurate quantitative glycoproteomics. Copyright © 2013 John Wiley & Sons, Ltd. |
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Keywords: | glycoproteomics label‐free quantitative proteomics glycopeptide ESI MALDI |
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