The analysis of N-linked glycans using liquid chromatography and mass spectrometry (LC-MS) presents significant challenges, particularly owing to their hydrophilic nature. To address these difficulties, a variety of derivatization methods has been developed to facilitate improved ionization and detection sensitivity. One such method, the Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT)™ strategy for labeling glycans, has previously been utilized in the analysis of Nand O-linked glycans in biological samples. To assess the maximum sensitivity and separability of the INLIGHT ™ preparation and analysis pipeline, several critical steps were investigated. First, recombinant and nonrecombinant sources of PNGase F were compared to assess variations in the released glycans. Second, modifications in the INLIGHT™ derivatization step were evaluated including temperature optimization, solvent composition changes, and reaction condition length and tag concentration. Optimization of the modified method resulted in 20-100 times greater peak areas for the detected N-linked glycans in fetuin and horseradish peroxidase compared to the standard method. Furthermore, the identification of low abundance glycans, such as (Fuc) 1 (Gal) 2 (GlcNAc) 4 (Man) 3 (NeuAc) 1 and (Gal) 3 (GlcNAc) 5 (Man) 3 (NeuAc) 3, was possible. Finally, the optimal LC setup for the INLIGHT™ Terms of use and reuse: academic research for non-commercial purposes, see here for full terms. http://www.springer.com/gb/openaccess/authors-rights/aam-terms-v1

Glycans are responsible for many biological activities; however, their structures are incredibly diverse and complex, often rendering the field of glycomics unsolvable by a single analytical technique. The development of multiple chemical derivatization strategies and bioinformatic software is responsible for some of the greatest analytical gains in the field of glycomics. The INLIGHT strategy is a chemical derivatization technique using hydrazide chemistry to derivatize the reducing end of Nlinked glycans and incorporates either a natural (NAT, 12 C 6 ) or a stable-isotope label (SIL, 13 C 6 ) to carry out relative quantification. Here we present GlycoHunter, a user-friendly software created in MATLAB that enables researchers to accurately and efficiently process MS1 glycomics data where a NAT and SIL pair is generated for relative quantification, including but not limited to, INLIGHT. GlycoHunter accepts the commonly used data file formats imzML and mzXML and effectively identifies all peak pairs associated with NATand SIL-labeled N-linked glycans using MS1 data. It also includes the ability to tailor the search parameters and export the results for further analysis using Skyline or Excel.

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Glycosylation is a ubiquitous co-and post-translational modification involved in the sorting, folding, and trafficking of proteins in biological systems; in humans, >50% of gene products are glycosylated with the cellular machinery of glycosylation compromising~2% of the genome. Perturbations in glycosylation have been implicated in a variety of diseases including neurodegenerative diseases and certain types of cancer. However, understanding the relationship between a glycan and its biological role is often difficult due to the numerous glycan isomers that exist. To address this challenge, nanoflow liquid chromatography, ion mobility spectrometry, and mass spectrometry (nLC-IMS-MS) were combined with the Individuality Normalization when Labeling with the Isotopic Glycan Hydrazide Tags (INLIGHT™) strategy to study a series of glycan standards and those enzymatically released from the glycoproteins horseradish peroxidase, fetuin, and pooled human plasma. The combination of IMS and the natural (NAT) and stable-isotope label (SIL) in the INLIGHT™ strategy provided additional confidence for each glycan identification due to the mobility aligned NAT-and SIL-labeled glycans and further capabilities for isomer examinations. Additionally, molecular trend lines based on the IMS and MS dimensions were investigated for the INLIGHT™ derivatized glycans, facilitating rapid identification of putative glycans in complex biological samples.

Magnaporthe oryzae (M. oryzae) is a pathogenic, filamentous fungus that is a primary cause of rice blast disease. The M. oryzae protein MGG_13065, SCF E3 ubiquitin ligase complex F-box protein, has been identified as playing a crucial role in the infection process, specifically, as part of the ubiquitin mediated proteolysis pathway. Proteins targeted by MGG_13065 E3 ligase are first phosphorylated and then ubiquitinated by E3 ligase. In this study, we used a label-free quantitative global proteomics technique to probe the role of ubiquitination and phosphorylation in the mechanism of how E3 ligase regulates change in virulence of M. oryzae. To do this, we compared the WT M. oryzae 70-15 strain with a gene knock out (E3 ligase KO) strain. After applying a ≥ 5 normalized spectral count cutoff, a total of 4432 unique proteins were identified comprised of 4360 and 4372 in the WT and E3 ligase KO samples, respectively. Eighty proteins drastically increased in abundance, while 65 proteins decreased in abundance in the E3 ligase KO strain. Proteins (59) were identified only in the WT strain; 13 of these proteins had both phosphorylation and ubiquitination post-translational modifications. Proteins (71) were revealed to be only in the E3 ligase KO strain; 23 of the proteins have both phosphorylation and ubiquitination post-translational modifications. Several of these proteins were associated with key biological processes. These data greatly assist in the selection of future genes for functional studies and enable mechanistic insight related to virulence.