Quantitative mapping of glycoprotein micro-heterogeneity and macro-heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides Kathrin Stavenhagen, a Hannes Hinneburg, a Morten Thaysen-Andersen, b Laura Hartmann, a Daniel Varón Silva, a Jens Fuchser, c Stephanie Kaspar, c Erdmann Rapp, d Peter H. Seeberger a,e and Daniel Kolarich a * 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. Supporting information may be found in the online version of this article. Keywords: glycoproteomics; label-free quantitative proteomics; glycopeptide; ESI; MALDI Introduction Nature has developed numerous ways for linking sugars to pro- teins, providing cells with efficient tools to increase the functional dimensions of proteins without modifying their polypeptide se- quences or structures. Currently, there are more than 30 different types of linkages described carrying a variety of monosaccharides and/or oligosaccharides, increasing molecular diversity. [1,2] One of the best-studied types of protein glycosylation is protein N- glycosylation and O-glycosylation, and current estimations predict that 20–50% of human proteins are N-glycosylated. [3,4] Despite this overall number still being a matter of debate, it is generally accepted that glycoproteins channelled through the endoplasmic reticulum Golgi secretory pathway are almost exclusively subjected to N-glycosylation and/or O-glycosylation events. [5] For a large number of these secreted and membrane glycoproteins, a crucial functional involvement of their respective glycosylation has been shown in events such as cell–cell interactions, receptor recognition or protein half life. [6] In human immunoglobulin G, for instance, the addition of a single core fucose on its Fc N-glycans inhibits * Correspondence to: Daniel Kolarich, Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany. E-mail: daniel.kolarich@mpikg.mpg.de a Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany b Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney 2109 New South Wales, Australia c Bruker Daltonics, Bremen, Germany d Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany e Free University Berlin, Berlin, Germany J. Mass Spectrom. 2013, 48, 627–639 Copyright © 2013 John Wiley & Sons, Ltd. Special feature: perspective Received: 14 December 2012 Revised: 8 March 2013 Accepted: 24 March 2013 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/jms.3210 627