The Art of Destruction: Optimizing Collision Energies in Quadrupole-Time of Flight (Q-TOF) Instruments for Glycopeptide-Based Glycoproteomics

Abstract

In-depth site-specific investigations of protein glycosylation are the basis for understanding the biological function of glycoproteins. Mass spectrometry-based N- and O-glycopeptide analyses enable determination of the glycosylation site, site occupancy, as well as glycan varieties present on a particular site. However, the depth of information is highly dependent on the applied analytical tools, including glycopeptide fragmentation regimes and automated data analysis. Here, we used a small set of synthetic disialylated, biantennary N-glycopeptides to systematically tune Q-TOF instrument parameters towards optimal energy stepping collision induced dissociation (CID) of glycopeptides. A linear dependency of m/z-ratio and optimal fragmentation energy was found, showing that with increasing m/z-ratio, more energy is required for glycopeptide fragmentation. Based on these optimized fragmentation parameters, a method combining lower- and higher-energy CID was developed, allowing the online acquisition of glycan and peptide-specific fragments within a single tandem MS experiment. We validated this method analyzing a set of human immunoglobulins (IgA1+2, sIgA, IgG1+2, IgE, IgD, IgM) as well as bovine fetuin. These optimized fragmentation parameters also enabled software-assisted glycopeptide assignment of both N- and O-glycopeptides including information about the most abundant glycan compositions, peptide sequence and putative structures. Twenty-six out of 30 N-glycopeptides and four out of five O-glycopeptides carrying >110 different glycoforms could be identified by this optimized LC-ESI tandem MS method with minimal user input. The Q-TOF based glycopeptide analysis platform presented here opens the way to a range of different applications in glycoproteomics research as well as biopharmaceutical development and quality control.Graphical AbstractᅟElectronic supplementary materialThe online version of this article (doi:10.1007/s13361-015-1308-6) contains supplementary material, which is available to authorized users.

Flagellin Glycoproteomics of the Periodontitis Associated Pathogen Selenomonas sputigena Reveals Previously Not Described O-glycans and Rhamnose Fragment Rearrangement Occurring on the Glycopeptides

“…Collision-induced dissociation (CID) of glycopeptides usually results in a prominent Y1-ion that facilitates identification of the peptide-linked monosaccharide (22). Such Y1-assigments are, however, increasingly difficult if more than one site of glycosylation is present on a single peptide or if gas phase monosaccharide rearrangements occur (23). Halim and Zauner independently reported the detection of small product ion signals in some O-glycopeptide product ion spectra acquired from human urinary and human fibrinogen glycopeptides that they assigned to a hexose rearrangement (24,25).…”

Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

“…Systematic studies have investigated the glycopeptide (glycan and peptide) sequence coverage as a function of several variables e.g. the adduct formation, proton mobility and (normalized) collision energy in higher-energy collision dissociation (HCD) (110), beam-type (Q-TOF) CID (111)(112)(113), and the combined use of resonance activation (ion trap) CID and HCD (77). Together with conventional electron transfer dissociation (ETD), HCD and ion trap CID, which are often activated as "back-to-back" alternating dissociation events of the same isolated precursor ions or alternatively engaged in separate LC-MS/MS runs, form the most popular dissociation techniques because of their complementary nature when applied to N-glycopeptides.…”

Glycomic and glycoproteomic analysis of glycoproteins—a tutorial

“…The collision energy required for the optimal fragmentation of glycan and peptide portion of a glycopeptide differs considerably, and one recent technology termed collision energy stepping CID allows simultaneous acquisition of MS/MS spectra of glycopeptide at lower and higher collision energies [120]. …”