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.

Keywords: Collision energy stepping CID; Glycopeptide; Glycoproteomics; Immunoglobulin; N-glycan; O-glycan; Q-TOF; Synthetic glycopeptides.

Graphical Abstract

ᅟ

Figure 1

Collision energy optima for synthetic N-glycopeptides. For the peptide part, the intensity coverage was plotted versus the applied collision energy, and for the glycan part, the GlycoQuest score was used. From these plots, the respective optima were determined (refer to text for further details). For GP-M (left), the optimal collision energies were determined for charge states 2+, 3+, 4+, and 5+ (a) peptide part; (b) glycan part). Error bars represent the standard deviation determined from an average of approximately 180 individual product-ion spectra. Right: determination of the optimal collision energies for all three synthetic glycopeptides [charge 4+ (c) peptide part; (d) glycan part)]

Figure 2

Correlation between precursor m/z and optimal collision energies. Synthetic glycopeptides (diamonds) were spiked into a mixture of glycopeptides (circles) enriched from a tryptic digest derived from a complex sample and analyzed via C18-RP-LC-ESI-Q-TOF tandem MS. For the [M + 5H]⁵⁺ species and partially for the [M + 4H]⁴⁺ species of the synthetic glycopeptides, values were obtained additionally by direct infusion. The optimal collision energies for peptide backbone and glycan moiety were determined based on GlycoQuest Score and peptide intensity coverage. [M + 5H]⁵⁺ species are indicated in orange, [M + 4H]⁴⁺ in blue, [M + 3H]³⁺ in green, and [M + 2H]²⁺ in black

Figure 3

Representative spectra of automatically assigned N- and O-glycopeptides from IgD using the optimized Q-TOF collision energy stepping dissociation method. (a) tryptic N-glycopeptide TLLNASR (N367, [M + 3H]³⁺ at m/z 910.386) carrying a Hex₅HexNAc₅NeuAc N-glycan. The specific glycopeptide spectrum provided sufficient information to assign it as a monosialylated diantennary N-glycan with a bisecting GlcNAc. (b) Tryptic O-glycopeptide AQASSVPTAQPQAEGSLAK (Ser109, Ser110, Thr113) [M + 3H] ³⁺ at m/z 930.089 with a disialylated core 1 type glycan attached

Figure 4

Product-ion spectra of a manually identified IgA1 O-glycopeptide using the optimized Q-TOF collision energy stepping dissociation method. The glycopeptide [M + 5H]⁵⁺ 1146.0924 with 89His-Arg126 has several occupied O-glycosylation sites with a total composition of Hex₃HexNAc₄NeuAc. The glycopeptide could not be identified automatically because the peptide backbone also fragmented into b₁₁ and y₂₇ + glycan fragments. Diagnostic b-ion fragments of the y₂₇ initial fragment are highlighted in red. Note the specific, proline-rich nature of the peptide sequence, resulting in fragments with larger sequence gaps