Project description:The trypanosomatid protozoan parasite Leishmania has a significant impact on human health globally. Understanding the pathways associated with virulence within this significant pathogen is critical for identifying novel vaccination and chemotherapy targets. Within this study we leverage an ultradeep proteomic approach to improve our understanding of two virulence associated genes in Leishmania; the Golgi Mannose/Arabinopyranose/Fucose nucleotide-sugar transporter LPG2, and the mitochondrial fucosyltransferase FUT1. Using deep peptide fractionation followed by complementary fragmentation approaches with higher energy collisional dissociation (HCD) and Electron-transfer dissociation (ETD) allowed the identification of over 6500 proteins, nearly doubling the experimentally observed Leishmania major proteome. This deep proteomic analysis revealed significant quantitative differences in both lpg2- and fut1s mutants with FUT1-dependent changes linked to marked alterations within mitochondrial associated proteins while LPG2-dependent changes impacted multiple aspects of the secretory pathway. While FUT1 has been shown to fucosylate peptides in vitro, no evidence for protein fucosylation was identified within our ultradeep analysis nor did we observe fucosylated glycans within Leishmania glycopeptides isolated using HILIC enrichment. Combined this work provides a critical proteomic resource for the community on the observable Leishmania proteome as well as highlights phenotypic changes associated with LPG2/FUT1 which may guide the development of future therapeutics.
Project description:With the optional setting of multiple stepped collisional energies (NCEs), higher-energy collisional dissociation (HCD) as available on Orbitrap instruments is a widely adopted dissociation method for intact N-glycopeptide characterization, where peptide backbones and N-glycan moieties are selectively fragmented at high and low NCEs, respectively. Initially, a dependent setting of a central value plus minus a variation is available to the users to set up NCEs, and the combination of 30±10% to give the energies 20%/30%/40% has been mostly adopted in the literature. With the recent availability of independent NCE setup, we found that the combination of 20%/30%/30% is better than 20%/30%/40%; in the analysis of complex intact N-glycopeptides enriched from gastric cancer tissues, total IDs with spectrum-level FDR≤1%, site-specific IDs with site-determining fragment ions and structure-specific IDs with structure-diagnostic fragment ions were increased by 42% (4,767->6,746), 57% (599->942), and 97% (1771->3495), respectively. This finding will benefit all the coming N-glycoproteomics studies using HCD as the dissociation method.
Project description:Within the Burkholderia genus O-linked protein glycosylation is now known to be highly conserved at the pathway and glycosylation substrate levels. While inhibition of glycosylation has been shown to be detrimental to virulence in B. cenocepacia, little is known about the role of glycosylation in Burkholderia glycoproteins. Within this study we have sought to improve our understanding of the breadth and dynamics of the B. cenocepacia O-glycoproteome to identify glycoproteins which require glycosylation for functionality. Assessing the glycoproteome across multiple common culturing media (LB, TSB, and artificial sputum medium to simulate cystic fibrosis sputum-like conditions) we demonstrate at least 141 glycoproteins are subjected to glycosylation within B. cenocepacia K56-2. Leveraging this insight, we quantitively assessed the glycoproteome of B. cenocepacia using Data-Independent Acquisition (DIA) across culturing media and growth phases revealing most B. cenocepacia glycoproteins are express under all conditions. Examination of how the absence of glycosylation impacts the glycoproteome reveals only a subset of the glycoproteome (BCAL1086, BCAL2974, BCAL0525, BCAM0505 and BCAL0127) appear impacted by the loss of glycosylation. Assessing the proteomic and phenotypic impacts of the loss of these glycoproteins compared to glycosylation null strains revealing the loss of BCAL0525, and to a lesser extend BCAL0127, mirror the proteomic effects observed in the absence of glycosylation. Finally, we demonstrate the loss of glycosylation within BCAL0525 at Serine-358 results in both loss of motility and proteomic impacts on flagellar apparatus consistent with the loss of apparatus stability. Combined this work demonstrates that O-linked glycosylation of BCAL0525 is functionally important within B. cenocepacia.
Project description:We aimed to in-depth characterize and quantify salivary N-glycoproteomics. HILIC enrichment and LC-MS/MS were combined to investigate salivary glycosylation both at deglycopeptides level and glycopepeptides level, and alterations in site-specific glycoforms for human saliva were detected between lung cancer patients and healthy subjects. Firstly, intact glycopeptides were enriched from human saliva through using HILIC. Obtained intact glycopeptides were characterized by LC-MS/MS directly. Furthermore, the glycosylation sites were fully identified by LC-MS/MS followed by incubating with PNGase F. The developed workflow was applied to compare N-glycosites and intact N-glycopeptides in lung cancer group and healthy control group. Dysregulated N-glycosites as well as site-specific glycoforms were confidently observed in lung cancer and their potential value in clinical applications will be discussed.
Project description:The heterogeneity and complexity of glycosylation hinder the depth of site-specific glycoproteomics analysis. High-field asymmetric-waveform ion-mobility spectrometry (FAIMS) has shown to improve the scope of bottom-up proteomics. The benefits of FAIMS for quantitative N-glycoproteomics have not been investigated yet. In this work, we optimized FAIMS settings for N-glycopeptide identification, with or without the tandem mass tag (TMT) label. The optimized FAIMS approach significantly increased the identification of site-specific N-glycopeptides derived from the purified IgM protein or human lymphoma cells. We explored in detail the changes in FAIMS mobility caused by N-glycopeptides with different characteristics, including TMT labeling, charge state, glycan type, peptide sequence, glycan size and precursor m/z. Importantly, FAIMS also improved multiplexed N-glycopeptide quantification, both with the standard MS2 acquisition method and with our recently developed Glyco-SPS-MS3 method. The combination of FAIMS and Glyco-SPS-MS3 provided the highest quantitative accuracy and precision. Our results demonstrate the advantages of FAIMS for improved mass-spectrometry-based qualitative and quantitative N-glycoproteomics.
Project description:The global pandemic of severe acute pneumonia syndrome (COVID-19) caused by SARS-CoV-2 urgently calls for prevention and intervention strategies. The densely glycosylated spike (S) protein highly exposed on the viral surface is a determinant for virus binding and invasion into host cells as well as elicitation of a protective host immune response. Herein, we characterized the site-specific N-glycosylation of SARS-CoV-2 S protein using stepped collision energy (SCE) mass spectrometry (MS). Following digestion with two complementary proteases to cover all potential N-glycosylation sequons and integrated N-glycoproteomics analysis, we revealed the N-glycosylation profile of SARS-CoV-2 S proteins at the levels of intact N-glycopeptides and glycosites, along with the glycan composition and site-specific number of glycans. All 22 potential canonical N-glycosites were identified in S protein protomer. Of those, 18 N-glycosites were conserved between SARS-CoV and SARS-CoV-2 S proteins. Nearly all glycosites were preserved among the 753 SARS-CoV-2 genome sequences available in the public influenza database Global Initiative on Sharing All Influenza Data. By comparison, insect cell-expressed SARS-CoV-2 S protein contained 38 N-glycans, which were primarily assigned to the high-mannose type N-glycans, whereas the human cell-produced protein possessed up to 140 N-glycans largely belonging to the complex type. In particular, two N-glycosites located in the structurally exposed receptor-binding domain of S protein exhibited a relatively conserved N-glycan composition in human cells. This N-glycosylation profiling and determination of differences between distinct expression systems could shed light on the infection mechanism and promote development of vaccines and targeted drugs.
Project description:Protein glycosylation is a complex post-translational modification with crucial cellular functions in all domains of life. Currently, large-scale glycoproteomics approaches rely on glycan database dependent algorithms and are thus unsuitable for discovery-driven analyses of glycoproteomes. Therefore, we devised SugarPy, a glycan database independent Python module, and validated it on the glycoproteome of human breast milk. We further demonstrated its applicability by analyzing glycoproteomes with uncommon glycans stemming from the green algae Chalmydomonas reinhardtii and the archaeon Haloferax volcanii. Finally, SugarPy facilitated the novel characterization of glycoproteins from Cyanidioschyzon merolae.
Project description:While altered protein glycosylation is regarded a trait of oral squamous cell carcinoma (OSCC), its heterogeneous glycoproteome and dynamics with disease progression remain unmapped. Employing an integrated multi-omics approach comprising unbiased and quantitative glycomics and glycoproteomics on resected OSCC tissues, we firstly profiled the N-glycome that overall displayed uniform expression in OSCC patients with (N+, n = 19) and without (N0, n = 12) lymph node metastasis, suggesting relatively stable N-glycosylation during metastasis. Notably, glycoproteomics and advanced correlation analysis uncovered altered site-specific N-glycosylation and associations with clinicopathological features. Importantly, targeted analyses of the multi-omics data unveiled two N-glycans and three N-glycopeptides that were closely associated with patient survival. This study provides novel insight into the complex OSCC tissue N-glycoproteome forming an important resource to further explore the underpinning disease mechanisms and uncover new prognostic glyco-markers for OSCC.
Project description:Cancer stem cells (CSCs) are reported to be responsible for tumor initiation, progression, metastasis, and therapy resistance where P-glycoprotein (P-gp) as well as other glycoproteins are involved. Identification of these glycoprotein markers is critical for understanding the resistance mechanism and developing therapeutics. Here we report our comparative N-glycoproteomics study of MCF‐7/ADR vs. MCF-7 cells. With zic-HILIC enrichment, isotopic diethyl labeling, RPLC-MS/MS (HCD) analysis and GPSeeker DB search, differentially expressed N-glycosylation was quantitatively characterized at the intact N-glycopeptides levels.