DIALib: an automated ion library generator for data independent acquisition mass spectrometry analysis of peptides and glycopeptides

Abstract

Data Independent Acquisition (DIA) Mass Spectrometry (MS) workflows allow unbiased measurement of all detectable peptides from complex proteomes, but require ion libraries for interrogation of peptides of interest. These DIA ion libraries can be theoretical or built from peptide identification data from Data Dependent Acquisition (DDA) MS workflows. However, DDA libraries derived from empirical data rely on confident peptide identification, which can be challenging for peptides carrying complex post-translational modifications. Here, we present DIALib, software to automate the construction of peptide and glycopeptide Data Independent Acquisition ion Libraries. We show that DIALib theoretical ion libraries can identify and measure diverse N-and O-glycopeptides from yeast and mammalian glycoproteins without prior knowledge of the glycan structures present. We also show that DIALib libraries consisting only of glycan oxonium ions can quickly and easily provide a global glycosylation profile of the "oxoniome" of glycoproteomes. DIALib also enables the study of post-translational modifications beyond glycosylation, in isolation or combination, in complex proteomes from diverse biological and clinical samples.

OxoScan-MS: Oxonium ion scanning mass spectrometry facilitates plasma glycoproteomics in large scale

“…56,57 Recent studies have shown the utility of Y-type fragment ions for quantification and generation of site-specific glycopeptide information in DIA analysis. 36,38,58 Based on these observations, we developed a rolling collision energy scheme, such that the MS/MS spectra of each glycopeptide feature also contain useful Y-type fragments for targeted re-analysis.…”

“…Data-independent acquisition (DIA) methods, such as SWATH-MS, have been increasingly applied in the analysis of large proteomic sample series. [28][29][30][31] In glycoproteomics, DIA approaches have been applied to assess glycosite occupancy of enzymatically deglycosylated peptides [32][33][34][35] , and more recently, facilitated the post-acquisition analysis of intact glycopeptides, either by targeted extraction of abundant Y-type (intact peptide with glycan fragments of various sizes) ions [36][37][38][39][40] or by searching against spectral libraries. 15,[41][42][43] These approaches yield remarkable depth in comparative analyses and in generating spectral libraries, generally using higher-collisional dissociation (HCD) and/or electron-based fragmentation techniques.…”

“…[43][44][45] Precursor ion scanning and DIA methods have further been applied to quantify oxonium ions, small, singly-charged fragment ions ubiquitously found in glycopeptide HCD/CID MS/MS spectra [46][47][48] in biotherapeutics and purified glycoproteins, as well as complex biofluids. 36,39,[49][50][51][52][53][54] We herein focused on building on these technological advances for developing a glycoproteomic screening platform for high-throughput studies. In order to reach throughput at low cost, we take a two-step approach that separates glycopeptide quantification from sequence assignment.…”

The Role of Data-Independent Acquisition for Glycoproteomics

“…This oxonium ion profiling method represents an unbiased approach for a quick differentiation of multiglycosylated biotherapeutics or other glycoprotein samples. Using this approach, Phung et al (72) developed an automated ion library generator designated DIALib and profiled the oxoniomes in cell wall samples from wildtypes of yeast and its nine glycosylation mutants using the SWATH-based DIA approach. Note that, although the initial biosynthesis pathway and early processing steps before the production of Man 8 GlcNAc 2 glycan are identical in yeast and mammals, yeast tend to produce hypermannosylated glycans by adding additional mannoses (73).…”

Recent advancements in glycoproteomic studies: Glycopeptide enrichment and derivatization, characterization of glycosylation in SARS CoV2, and interacting glycoproteins

“…For data‐independent MS/MS acquisition (DIA) mode, a software termed DIALib was introduced to automate the creation of peptide and glycopeptide DIA ion libraries. DIALib's theoretical ion libraries were able to identify N‐ and O‐glycopeptides from yeast and mammalians without prior knowledge of their glycan structures (Phung, Zacchi, et al, 2020).…”