ABSTRACT
Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remains elusive. To this end, we have characterised the structure-biosynthesis-activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants and a previously unreported low-abundance monoprotomer. Longitudinal profiling of maturing, mature, granule-separated and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, molecular modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity.
Subject(s)
Cytoplasmic Granules/enzymology , Glycopeptides/metabolism , Neutrophils/enzymology , Peroxidase/metabolism , Polysaccharides/chemistry , Polysaccharides/metabolism , Glycopeptides/chemistry , Glycosylation , HumansABSTRACT
Invertases are glycosidases applied for synthesis of alkyl glycosides that are important and effective surfactants. Stability of invertases in the environment with increased content of organic solvent is crucial for increase of productivity of glycosidases. Their stability is significantly influenced by N-glycosylation. However, yeast N-glycosylation pathways may synthesize plethora of N-glycan structures. A total natural crude mixture of invertase glycoforms (EINV) extracted from Saccharomyces cerevisiae was subfractionated by anion-exchange chromatography on industrial monolithic supports to obtain different glycoforms (EINV1-EINV3). Separated glycoforms exhibited different stabilities in water-alcohol solutions that are in direct correlation with the amount of phosphate bound to N-glycans. Observed differences in stability of different invertase glycoforms were used to improve productivity of methyl ß-d-fructofuranoside (MF) synthesis. The efficiency and yield of MF synthesis were improved more than 50% when the most stabile glycoform bearing the lowest amount of phosphorylated N-glycans is selected and utilized. These data underline the importance of analysis of glycan structures attached to glycoproteins, demonstrate different impact of N-glycans on the surface charge and enzyme stability in regard to particular reaction environment, and provide a platform for improvement of yield of industrial enzymatic synthesis by chromatographic selection of glycoforms on monolithic supports.
Subject(s)
Saccharomyces cerevisiae , beta-Fructofuranosidase , Chromatography, Ion Exchange , Glycosylation , Polysaccharides , Saccharomyces cerevisiae/enzymologyABSTRACT
Protein glycosylation impacts the development and function of innate immune cells. The glycophenotypes and the glycan remodelling associated with the maturation of macrophages from monocytic precursor populations remain incompletely described. Herein, label-free porous graphitised carbon-liquid chromatography-tandem mass spectrometry (PGC-LC-MS/MS) was employed to profile with high resolution the N- and O-glycome associated with human monocyte-to-macrophage transition. Primary blood-derived CD14+ monocytes were differentiated ex vivo in the absence of strong anti- and proinflammatory stimuli using a conventional 7-day granulocyte-macrophage colony-stimulating factor differentiation protocol with longitudinal sampling. Morphology and protein expression monitored by light microscopy and proteomics validated the maturation process. Glycomics demonstrated that monocytes and macrophages display similar N-glycome profiles, comprising predominantly paucimannosidic (Man1-3GlcNAc2Fuc0-1, 22.1-30.8%), oligomannosidic (Man5-9GlcNAc2, 29.8-35.7%) and α2,3/6-sialylated complex-type N-glycans with variable core fucosylation (27.6-39.1%). Glycopeptide analysis validated conjugation of these glycans to human proteins, while quantitative proteomics monitored the glycoenzyme expression levels during macrophage differentiation. Significant interperson glycome variations were observed suggesting a considerable physiology-dependent or heritable heterogeneity of CD14+ monocytes. Only few N-glycome changes correlated with the monocyte-to-macrophage transition across donors including decreased core fucosylation and reduced expression of mannose-terminating (paucimannosidic-/oligomannosidic-type) N-glycans in macrophages, while lectin flow cytometry indicated that more dramatic cell surface glycan remodelling occurs during maturation. The less heterogeneous core 1-rich O-glycome showed a minor decrease in core 2-type O-glycosylation but otherwise remained unchanged with macrophage maturation. This high-resolution glycome map underpinning normal monocyte-to-macrophage transition, the most detailed to date, aids our understanding of the molecular makeup pertaining to two vital innate immune cell types and forms an important reference for future glycoimmunological studies.
Subject(s)
Macrophages/metabolism , Monocytes/metabolism , Polysaccharides/metabolism , Chromatography, Liquid , Glycomics , Glycopeptides/analysis , Glycosylation , Humans , Polysaccharides/chemistry , Tandem Mass SpectrometryABSTRACT
While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics-centric study investigates a possible link between protein paucimannosylation, an under-studied class of human N-glycosylation [Man1-3 GlcNAc2 Fuc0-1 ], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non-cancerous specimens are profiled from 467 published and unpublished PGC-LC-MS/MS N-glycome datasets collected over a decade. PMGs, particularly Man2-3 GlcNAc2 Fuc1 , are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0-50.2%). Analyses of paired (tumor/non-tumor) and stage-stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N-acetyl-ß-hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.
Subject(s)
Mannose/metabolism , Neoplasms/metabolism , Cell Line, Tumor , Chromatography, Liquid , Disease Progression , Glycosylation , Humans , Tandem Mass SpectrometryABSTRACT
Deep characterization of biologically relevant glycans remains challenging. Porous graphitized carbon-liquid chromatography tandem mass spectrometry (PGC-LC-MS/MS) enables the quantitative elucidation of glycan fine structures. However, the early PGC-LC elution of smaller glycans (tri-, tetra-, and pentasaccharides) at low organic solvent content hampers their detection. In efforts to improve the glycan profiling sensitivity and accuracy, we present a new capillary-flow PGC-LC-MS/MS-based configuration comprising a post-column make-up flow (PCMF) that supplies an ion-promoting organic solvent to separated glycans prior to their detection by MS. The analytical performance of this setup was systematically evaluated against our existing capillary-flow PGC-LC-MS/MS platform (Jensen et al., Nat. Protoc. 2012, 7, 1299). Specifically, the ion intensities and signal-to-noise ratios of various classes of nonderivatized glycans from N- and O-glycoproteins and fructooligosaccharide mixtures were compared using methanol (MeOH)-, isopropanol (IPA)-, and acetonitrile (ACN)-based PCMF at various concentrations. In particular, ACN- and IPA-based PCMF dramatically increased the signal response across all glycan types (30- to 100-fold), improved the MS/MS spectral quality, and reduced the quantitative glycoprofile variation between replicates. In particular, the detection of the early eluting glycans benefitted from the PCMF. The highest sensitivity gains were achieved with the supplements of 100% ACN and IPA (equating to 57% (v/v) net concentration at the ion source) while neither compromising the favorable PGC-LC properties including the high peak capacity and glycan isomer separation nor changing the MS detection behavior. In conclusion, PCMF-based PGC-LC-MS/MS dramatically improves the glycomics sensitivity, coverage, and quantitative accuracy not least for the difficult-to-detect early eluting and low-abundance glycans detached from N- and O-glycoproteins.
Subject(s)
Chromatography, High Pressure Liquid/methods , Glycomics/methods , Polysaccharides/analysis , 2-Propanol/chemistry , Acetonitriles/chemistry , Carbon , Glycoproteins/chemistry , Isomerism , Porosity , Tandem Mass SpectrometryABSTRACT
We established a small synthetic N-glycopeptide library to systematically evaluate the effect of glycosylation site location and glycan size on the efficiency of electron transfer dissociation (ETD) fragmentation and subsequent automated identification. The glycopeptides within this library differed in glycosylation site position and glycan size ranging from the pentasaccharide N-glycan core to fully sialylated, biantennary N-glycans. Factors such as glycan size, glycosylation site position within a glycopeptide and individual precursor m/z all significantly impacted the number and quality of assignable glycopeptide backbone fragments. Generally, high charge/low m/z precursors (>3+) and glycopeptides carrying neutral, smaller N-glycans gave better product ion spectra, while hardly any product ions were detectable for sialylated, triply charged N-glycopeptides. These factors impacted correct glycopeptide identification by proteomics software tools such as SEQUEST or Amanda. A better understanding how glycopeptide physico-chemical properties influence fragmentation will help optimizing fragmentation conditions and generate better data, which will facilitate software assisted glycopeptide data analyses.
Subject(s)
Amino Acid Sequence/genetics , Glycopeptides/chemistry , Polysaccharides/chemistry , Proteomics , Electron Transport/genetics , Electrons , Glycopeptides/genetics , Glycosylation , Humans , Ions/chemistry , Polysaccharides/genetics , Software , Tandem Mass SpectrometryABSTRACT
N- and O-glycans are attractive clinical biomarkers as glycosylation changes in response to diseases. The limited availability of defined clinical specimens impedes glyco-biomarker identification and validation in large patient cohorts. Formalin-fixed paraffin-embedded (FFPE) clinical specimens are the common form of sample preservation in clinical pathology, but qualitative and quantitative N- and O-glycomics of such samples has not been feasible to date. Here, we report a highly sensitive and glycan isomer selective method for simultaneous N- and O-glycomics from histopathological slides. As few as 2000 cells isolated from FFPE tissue sections by laser capture microdissection were sufficient for in-depth histopathology-glycomics using porous graphitized carbon nanoLC ESI-MS/MS. N- and O-glycan profiles were similar between unstained and hematoxylin and eosin stained FFPE samples but differed slightly compared with fresh tissue. This method provides the key to unlock glyco-biomarker information from FFPE histopathological tissues archived in pathology laboratories worldwide.
Subject(s)
Biomarkers/metabolism , Glycomics/methods , Laser Capture Microdissection/methods , Neoplasms/metabolism , Chromatography, Liquid/methods , Humans , Paraffin Embedding , Polysaccharides/analysis , Polysaccharides/chemistry , Tandem Mass Spectrometry/methods , Tissue FixationABSTRACT
Ants (Hymenoptera: Formicidae) are familiar inhabitants of most terrestrial environments. Although we are aware of the ability of many species to sting, knowledge of ant venom chemistry remains limited. Herein, we describe the discovery and characterization of an O-linked glycopeptide (Mg7a) as a major component of the venom of the ant Myrmecia gulosa. Electron transfer dissociation and higher-energy collisional dissociation tandem mass spectrometry were used to localize three α-N-acetylgalactosaminyl residues (α-GalNAc) present on the 63-residue peptide. To allow for functional studies, we synthesized the full-length glycosylated peptide via solid-phase peptide synthesis, combined with diselenide-selenoester ligation-deselenization chemistry. We show that Mg7a is paralytic and lethal to insects, and triggers pain behavior and inflammation in mammals, which it achieves through a membrane-targeting mode of action. Deglycosylation of Mg7a renders it insoluble in aqueous solution, suggesting a key solubilizing role of the O-glycans.
ABSTRACT
The vast heterogeneity of protein glycosylation, even of a single glycoprotein with only one glycosylation site, can give rise to a set of macromolecules with different physicochemical properties. Thus, the use of orthogonal approaches for comprehensive characterization of glycoproteins is a key requirement. This chapter describes a universal workflow for site-specific N- and O-glycopeptide analysis. In a first step glycoproteins are treated with Pronase to generate glycopeptides containing small peptide sequences for enhanced glycosylation site assignment and characterization. These glycopeptides are then separated and detected using an integrated C18-porous graphitized carbon-liquid chromatography (PGC-LC) setup online coupled to a high-resolution electrospray ionization (ESI)-quadrupole time-of-flight (QTOF)-mass spectrometer operated in a combined higher- and lower-energy CID (stepping-energy CID) mode. The LC-setup allows retention of more hydrophobic glycopeptides on C18 followed by subsequent capturing of C18-unbound (glyco)peptides by a downstream placed PGC stationary phase. Glycopeptides eluted from both columns are then analyzed within a single analysis in a combined data acquisition mode. Stepping-energy CID results in B- and Y-ion fragments originating from the glycan moiety as well as b- and y-ions derived from the peptide part. This allows simultaneous site-specific identification of the glycan and peptide sequence of a glycoprotein.
Subject(s)
Chromatography, Liquid/methods , Glycopeptides/analysis , Glycoproteins/chemistry , Graphite/chemistry , Polysaccharides/analysis , Spectrometry, Mass, Electrospray Ionization/methods , Alkylation , Electrophoresis, Polyacrylamide Gel/methods , Glycomics/methods , Humans , Porosity , Proteomics/methods , Tandem Mass Spectrometry/methodsABSTRACT
The availability of well-defined samples in sufficient numbers represents a major bottleneck for any biomarker related research. The utilization of preserved, archived and clinically well-described samples therefore holds a great potential to bridge this gap. This chapter describes a universal workflow for the comprehensive characterization of N- and O-glycans released from whole formalin-fixed, paraffin-embedded tissue sections, including an option for further partitioning using laser microdissection of specific tissue areas/cell populations. Glycoproteins are extracted and subsequently immobilized onto a PVDF membrane prior enzymatic release of N-glycans. Following N-glycan retrieval O-glycans are released using reductive ß-elimination from the same sample spot, significantly reducing the required amount of starting material. Released and reduced glycan structures are characterized using porous graphitized carbon liquid chromatography online coupled to an electrospray ionization-ion trap mass spectrometer. This technique provides information on the relative abundances of individual glycans along with detailed structural information, including isomer differentiation and functional epitope characterization of N- and O-glycans obtained from minimal amounts of tissue down to a few thousand cells.
Subject(s)
Glycomics/methods , Glycoproteins/chemistry , Paraffin Embedding/methods , Polysaccharides/analysis , Tissue Fixation/methods , Animals , Chromatography, Liquid/methods , Formaldehyde/chemistry , Graphite/chemistry , Humans , Immobilized Proteins/chemistry , Laser Capture Microdissection/methods , Membranes, Artificial , Proteomics/methods , Spectrometry, Mass, Electrospray Ionization/methods , Staining and Labeling/methods , Tandem Mass Spectrometry/methodsABSTRACT
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.
Subject(s)
Fetuins/chemistry , Glycopeptides/analysis , Immunoglobulins/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Amino Acid Sequence , Animals , Carbohydrate Sequence , Cattle , Humans , Proteomics/methods , Tandem Mass Spectrometry/methodsABSTRACT
Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached to particular protein glycosylation sites (micro-heterogeneity) and evaluate the molar site occupancy (macro-heterogeneity) in glycoproteomics. However, the accuracy of MS for such quantitative measurements remains to be clarified. As a key step towards this goal, a panel of related tryptic peptides with and without complex, biantennary, disialylated N-glycans was chemically synthesised by solid-phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A and endo D/F/H were synthetically produced, carrying aspartic acid and N-acetylglucosamine-linked asparagine residues, respectively, at the glycosylation site. The MS ionisation/detection strengths of these pure, well-defined and quantified compounds were investigated using various MS ionisation techniques and mass analysers (ESI-IT, ESI-Q-TOF, MALDI-TOF, ESI/MALDI-FT-ICR-MS). Depending on the ion source/mass analyser, glycopeptides carrying complex-type N-glycans exhibited clearly lower signal strengths (10-50% of an unglycosylated peptide) when equimolar amounts were analysed. Less ionisation/detection bias was observed when the glycopeptides were analysed by nano-ESI and medium-pressure MALDI. The position of the glycosylation site within the tryptic peptides also influenced the signal response, in particular if detected as singly or doubly charged signals. This is the first study to systematically and quantitatively address and determine MS glycopeptide ionisation/detection strengths to evaluate glycoprotein micro-heterogeneity and macro-heterogeneity by label-free approaches. These data form a much needed knowledge base for accurate quantitative glycoproteomics.