Figure - available from: Mass Spectrometry Reviews
This content is subject to copyright. Terms and conditions apply.
![Schematic of two‐step derivatization of sialic acid linkage isomers. (A) α2,6‐linked sialic acid forms a stable dimethylamide through EDC, HOBt, and dimethylamine reactions in the first step, and keeps the same structure in the second step. (B) α2,3‐linked sialic acid forms an unstable lactone in the first step, and then convert to a stable amide in the second step. (C) Mass spectra of in situ derivatized (top) and native (bottom) N‐glycans derived from leiomyosarcoma FFPE tissue showing the induced mass shift of +28.031 Da between α2,3‐ and α2,6‐linked sialic acids after derivatization, while native N‐glycans are detected with additional neutral proton‐sodium exchange. Symbols: green circle represents mannose, yellow circle represents galactose (Gal), blue square represents N‐acetylglucosamine (GlcNAc), yellow square represents N‐acetylgalactosamine (GalNAc), red triangle represents fucose, purple diamond represents N‐acetylneuraminic acid (NeuAc). Reproduced with the permission from Holst et al. (2016) [Color figure can be viewed at wileyonlinelibrary.com]](publication/352682003/figure/fig1/AS:11431281115740040@1675090952070/Schematic-of-two-step-derivatization-of-sialic-acid-linkage-isomers-A-a2-6-linked.png)
Schematic of two‐step derivatization of sialic acid linkage isomers. (A) α2,6‐linked sialic acid forms a stable dimethylamide through EDC, HOBt, and dimethylamine reactions in the first step, and keeps the same structure in the second step. (B) α2,3‐linked sialic acid forms an unstable lactone in the first step, and then convert to a stable amide in the second step. (C) Mass spectra of in situ derivatized (top) and native (bottom) N‐glycans derived from leiomyosarcoma FFPE tissue showing the induced mass shift of +28.031 Da between α2,3‐ and α2,6‐linked sialic acids after derivatization, while native N‐glycans are detected with additional neutral proton‐sodium exchange. Symbols: green circle represents mannose, yellow circle represents galactose (Gal), blue square represents N‐acetylglucosamine (GlcNAc), yellow square represents N‐acetylgalactosamine (GalNAc), red triangle represents fucose, purple diamond represents N‐acetylneuraminic acid (NeuAc). Reproduced with the permission from Holst et al. (2016) [Color figure can be viewed at wileyonlinelibrary.com]
Source publication
Glycosylation is one of the most significant and abundant posttranslational modifications in mammalian cells. It mediates a wide range of biofunctions, including cell adhesion, cell communication, immune cell trafficking, and protein stability. Also, aberrant glycosylation has been associated with various diseases such as diabetes, Alzheimer's dise...
Similar publications
N-glycosylation is one of the most abundant post-translational modifications of proteins, essential for many physiological processes, including protein folding, protein stability, oligomerization and aggregation, and molecular recognition events. Defects in the N-glycosylation pathway cause diseases that are classified as congenital disorders of gl...
Citations
... [1][2][3] The study of glycoproteins has long been a fast-growing field of scientific endeavor and is still attracting significant research interest, which is not surprising since it is estimated that more than 50% of mammalian proteins are attached to oligosaccharide chains ("glycans"). 4 The numerous studies exploring genetic and cellular aspects of glycosylation processes have demonstrated the fundamental significance of glycoproteins, thus leading glycosciences into the spotlight of modern biomedical research. 5,6 As a result of their high abundance, it is clear that glycoproteins play an important role in numerous biological functions, including immunological responses, cell adhesion, intra and intercellular signaling, cell−matrix interactions, tumor progression and metastasis, and protein stability. ...
... Consequently, exploring appropriate fragmentation techniques is critical for reliable glycopeptide identification. 4,134 Characterization of glycopeptides using LC-MS/MS LC analysis of glycopeptides is frequently employed using nano-LC-MS/MS systems, whereby eluted glycopeptides from the LC column are subjected to ionization using ESI and fragmented by a variety of tandem MS sophisticated fragmentation methods, such as collision, electron, or photo-induced fragmentations. 31,89,135,136 Due to the availability of various stationary phases, such as reversed-phase liquid chromatography (RP-LC), HILIC, porous graphitized carbon (PGC), and ion-pairing chromatography, this technique has emerged as a prevailing tool in glycoproteomics. ...
... MALDI is a vaporization-ionization technique commonly employed for the analysis of complex biomolecules like peptides and protein mixtures. 4,35,168 In this method, the sample is combined with an appropriate organic matrix solution and then allowed to co-crystallize directly on specialized sample plates. MALDI has found extensive biological applications in glycomics and glycoproteomics, particularly in profiling released glycans derived from glycoproteins in biological mixtures. ...
Protein glycosylation, a highly significant and ubiquitous post-translational modification (PTM) in eukaryotic cells, has attracted considerable research interest due to its pivotal role in a wide array of essential biological processes. Conducting a comprehensive analysis of glycoproteins is imperative for understanding glycoprotein bio-functions and identifying glyco-sylated biomarkers. However, the complexity and heterogeneity of glycan structures, coupled with the low abundance and poor ionization efficiencies of glycopeptides have all contributed to making the analysis and subsequent identification of glycans and glycopeptides much more challenging than any other biopolymers. Nevertheless, the significant advancements in enrichment techniques, chromatographic separation, and mass spectrometric methodologies represent promising avenues for mitigating these challenges. Numerous substrates and multifunctional materials are being designed for glycopeptide enrichment, proving valuable in glycomics and glycoproteomics. Mass spectrometry (MS) is pivotal for probing protein glycosylation, offering sensitivity and structural insight into glycopeptides and glycans. Additionally, enhanced MS-based glycopeptide characterization employs various separation techniques like liquid chromatography, capillary electrophoresis, and ion mobility. In this review, we highlight recent advances in enrichment methods and MS-based separation techniques for analyzing different types of protein glycosylation. This review also discusses various approaches employed for glycan release that facilitate the investigation of the glycosylation sites of the identified glycoproteins. Furthermore, numerous bioinformatics tools aiding in accurately characterizing glycan and glycopeptides are covered.
... Glycosylated proteins, known as glycoproteins, account for over 50% of all mammalian proteins [9]. Given the significant role of glycoproteins in vital biological processes [10] and the correlation of their altered expressions with various diseases [11][12][13][14] and cancers [15][16][17], reliable quantitative and qualitative glycoproteomics methodologies have become increasingly critical for understanding these protein modification sites [18,19]. ...
Glycosylation of proteins is an important post-translational modification that plays a role in a wide range of biological processes, including immune response, intercellular signaling, inflammation, and host-pathogen interaction. Abnormal protein glycosylation has been correlated with various diseases. However, the study of protein glycosylation remains challenging due to its low abundance, microheterogeneity of glycosylation sites, and low ionization efficiency. During the past decade, several methods for enrichment and for isolation of glycopeptides from biological samples have been developed and successfully employed in glycoproteomics research. In this chapter, we discuss the sample preparation protocol and the strategies for effectively isolating and enriching glycopeptides from biological samples, using PolyHYDROXYETHYL A as a hydrophilic interaction liquid chromatography (HILIC) enrichment technique.
... Most commonly used software for glycopeptide identification [158]. ...
Glycosylation, the major post‐translational modification of proteins, significantly increases the diversity of proteoforms. Glycans are involved in a variety of pivotal structural and functional roles of proteins, and changes in glycosylation are profoundly connected to the progression of numerous diseases. Mass spectrometry (MS) has emerged as the gold standard for glycan and glycopeptide analysis because of its high sensitivity and the wealth of fragmentation information that can be obtained. Various separation techniques have been employed to resolve glycan and glycopeptide isomers at the front end of the MS. However, differentiating structures of isobaric and isomeric glycopeptides constitutes a challenge in MS‐based characterization. Many reports described the use of various ion mobility–mass spectrometry (IM–MS) techniques for glycomic analyses. Nevertheless, very few studies have focused on N ‐ and O ‐linked site‐specific glycopeptidomic analysis. Unlike glycomics, glycoproteomics presents a multitude of inherent challenges in microheterogeneity, which are further exacerbated by the lack of dedicated bioinformatics tools. In this review, we cover recent advances made towards the growing field of site‐specific glycosylation analysis using IM–MS with a specific emphasis on the MS techniques and capabilities in resolving isomeric peptidoglycan structures. Furthermore, we discuss commonly used software that supports IM–MS data analysis of glycopeptides.
... N-Glycan microheterogeneity is another factor that adds complexity to the study of N-glycosylation. The N-glycans can present isomeric modifications in different structural moieties, such as the sialic acid, mannose, or fucose units [28,53]. Thus, as a glycan increases in size, its structural complexity also increases. ...
The SARS-CoV-2 virus rapidly spread worldwide, threatening public health. Since it emerged, the scientific community has been engaged in the development of effective therapeutics and vaccines. The subunit S1 in the spike protein of SARS-CoV-2 mediates the viral entry into the host and is therefore one of the major research targets. The S1 protein is extensively glycosylated, and there is compelling evidence that glycans protect the virus’ active site from the human defense system. Therefore, investigation of the S1 protein glycome alterations in the different virus variants will provide a view of the glycan evolution and its relationship with the virus pathogenesis. In this study, we explored the N-glycosylation expression of the S1 protein for eleven SARS-CoV-2 variants: five variants of concern (VOC), including alpha, beta, gamma, delta, and omicron, and six variants of interest (VOI), including epsilon, eta, iota, lambda, kappa, and mu. The results showed significant differences in the N-glycome abundance of all variants. The N-glycome of the VOC showed a large increase in the abundance of sialofucosylated glycans, with the greatest abundance in the omicron variant. In contrast, the results showed a large abundance of fucosylated glycans for most of the VOI. Two glycan compositions, GlcNAc4,Hex5,Fuc,NeuAc (4-5-1-1) and GlcNAc6,Hex8,Fuc,NeuAc (6-8-1-1), were the most abundant structures across all variants. We believe that our data will contribute to understanding the S1 protein’s structural differences between SARS-CoV-2 mutations.
... -Sialik asit ile modifiye edilmiş glikanların analizi -N-glikan karakterizasyonu HILIC-MS [166,167] -Glikanlar yüksek derecede hidrofilik olduklarından, HILIC-MS, doğal ve indirgeyici uç etiketli glikanları analiz etmek için en iyi yöntemlerden biri olarak kabul edilmektedir. ...
Glikanlar, çeşitli biyolojik süreçlerde önemli rol oynayan ve sağlık ile hastalık üzerinde önemli etkileri olan karmaşık karbonhidrat molekülleri olarak bilinmektedir. Glikanların kapsamlı bir şekilde analiz edilmesi, gelişmiş analitik tekniklerin bir kombinasyonunu gerektirmektedir. Bu derleme, glikan analizinde kullanılan çeşitli tekniklerin, örnekleme hazırlığı, glikan zenginleştirme, glikan salımı, etiketleme, ayrıştırma ve tespit gibi adımlarının ayrıntılı bir iş akışını sunmaktadır. Her adımın prensipleri, uygulamaları ve avantajları açıklanarak, glikan araştırmalarına katkıları vurgulanmaktadır. Ayrıca, spesifik glikan analiz hedefleri için uygun tekniklerin seçiminin önemi üzerinde durulmaktadır. Bu iş akışı, glikanların kapsamlı bir anlayışını sağlayarak, biyolojik sistemlerdeki rollerini açığa çıkarmaya ve yeni terapötik müdahalelerin geliştirilmesine yardımcı olmaktadır.
... These spectra result from two or more precursors being selected in the same MS/MS event and complicate the process of spectra matching and glycopeptide identification [7]. In this sense, the use of ion mobility (IM) is a promising additional tool for isomer separation [41]. It can be considered an additional separation dimension, where ions are separated based on their collisional cross-sections. ...
... Assigning signals to individual structures, with detailed description of stereochemistry, is not straightforward in large-scale investigations. Reasons that hinder comprehensive glycoproteomics isomeric analysis are connected with IM technology, which has not sufficient resolution to achieve complete separation of isomers in complex biological samples or complex structural molecules [41]. The use of collisional cross-sections, as additional criteria for structural identification by database search, also has limitations that prevent application in routine identification. ...
This trends article provides an overview of the state of the art in the analysis of intact glycopeptides by proteomics technologies based on LC–MS analysis. A brief description of the main techniques used at the different steps of the analytical workflow is provided, giving special attention to the most recent developments. The topics discussed include the need for dedicated sample preparation for intact glycopeptide purification from complex biological matrices. This section covers the common approaches with a special description of new materials and innovative reversible chemical derivatization strategies, specifically devised for intact glycopeptide analysis or dual enrichment of glycosylation and other post-translational modifications. The approaches are described for the characterization of intact glycopeptide structures by LC–MS and data analysis by bioinformatics for spectra annotation. The last section covers the open challenges in the field of intact glycopeptide analysis. These challenges include the need of a detailed description of the glycopeptide isomerism, the issues with quantitative analysis, and the lack of analytical methods for the large-scale characterization of glycosylation types that remain poorly characterized, such as C-mannosylation and tyrosine O-glycosylation. This bird’s-eye view article provides both a state of the art in the field of intact glycopeptide analysis and open challenges to prompt future research on the topic.
Graphical Abstract
... Recently, researchers have found great potential in employing glycomics to identify diagnostic biomarkers in circulating biofluids for brain-related diseases [12][13][14][15][16][17][18][19][20]. Additionally, it is well known that glycans have a variety of isomeric conformations where some of the most important structures have differences in the sialic acid linkage (e.g., 2,3/2, 6), fucosylation in the glycan core or branch, and the formation of bisecting Nacetylglucosamine in the glycan core [13,21,22]. Glycan isomers were found to be associated with numerous diseases [12,[23][24][25] and various cancers [26][27][28]. ...
Mild cognitive impairment (MCI) is an early stage of memory loss that affects cognitive abilities, such as language or virtual/spatial comprehension. This cognitive decline is mostly observed with the aging of individuals. Recently, MCI has been considered as a prodromal phase of Alzheimer’s disease (AD), with a 10–15% conversion rate. However, the existing diagnostic methods fail to provide precise and well-timed diagnoses, and the pathophysiology of MCI is not fully understood. Alterations of serum N-glycan expression could represent essential contributors to the overall pathophysiology of neurodegenerative diseases and be used as a potential marker to assess MCI diagnosis using non-invasive procedures. Herein, we undertook an LC-MS/MS glycomics approach to determine and characterize potential N-glycan markers in depleted blood serum samples from MCI patients. For the first time, we profiled the isomeric glycome of the low abundant serum glycoproteins extracted from serum samples of control and MCI patients using an LC-MS/MS analytical strategy. Additionally, the MRM validation of the identified data showed five isomeric N-glycans with the ability to discriminate between healthy and MCI patients: the sialylated N-glycans GlcNAc5,Hex6,Neu5Ac3 and GlcNAc6,Hex7,Neu5Ac4 with single AUCs of 0.92 and 0.87, respectively, and a combined AUC of 0.96; and the sialylated-fucosylated N-glycans GlcNAc4,Hex5,Fuc,Neu5Ac, GlcNAc5,Hex6,Fuc,Neu5Ac2, and GlcNAc6,Hex7,Fuc,Neu5Ac3 with single AUCs of 0.94, 0.67, and 0.88, respectively, and a combined AUC of 0.98. According to the ingenuity pathway analysis (IPA) and in line with recent publications, the identified N-glycans may play an important role in neuroinflammation. It is a process that plays a fundamental role in neuroinflammation, an important process in the progression of neurodegenerative diseases.
... This analytical challenge has long been tackled using a variety of glycomics techniques, which all profile different aspects of glycosylation and exhibit specific analytical advantages and disadvantages. Importantly however, the use of this wide range of methodologies, including lectin-arrays 16 , MALDI-TOF MS analysis of underivatized 17 , permethylated 18 or derivatized glycans 19,20 , fluorescence labelling of glycans followed by either reversed-or normal-phase HPLC analysis 21,22 , to various LC-MS and LC-MS/MS analysis workflows 23,24 , leads to low comparability between studies. ...
Over 50% of all human proteins are post-translationally modified by glycans, which alter their functions in fundamental biological processes. Mice (mus musculus) represent the most common mammalian model organism to study fundamental biological processes, including glycosylation. Because of isomeric variance, the comprehensive analysis of protein-linked N-glycans is a challenging task. Coupled with high-resolution mass-spectrometry, porous graphitic carbon (PGC)-LC is a powerful and widely used method for isomer-specific glycan analysis. Here we report on a consistent and complete PGC-LC-MS/MS N-glycome dataset of 23 different mouse tissues, critically complementing existing glycan data-repositories. We used PGC-LC to chromatographically separate even closely related N-glycan isomers and an Orbitrap Exploris 480 mass-spectrometer for data-acquisition. Multivariate data analysis revealed tissue specific N-glycome signatures, highlighted organ-intrinsic regulations of glycobiological pathways and confirmed prior glycobiological knowledge, as exemplified by the brain N-glycome. This dataset can be used for fundamental glycobiological research, glycan analytical benchmarking, the development of new mass-spectrometric data analysis tools, glycobiological pathway modelling and simulation, as well as for integrative systems biology.
... MS methods require significantly reduced sample quantities (micromolar to nanomolar concentrations) compared to NMR and X-ray crystallography (Mechref & Novotny, 2002). Additionally, because MS monitors the mass-tocharge (m/z) ratio of analytes, heterogeneous structures with different numbers of monosaccharides can be distinguished by differences in m/z, while isomers can be characterized with tandem methods, including ion mobility spectrometry (IMS)-MS and tandem MS (MS/ MS) (Grabarics et al., 2021;Peng et al., 2021). ...
Glycans, carbohydrates, and glycoconjugates are involved in many crucial biological processes, such as disease development, immune responses, and cell–cell recognition. Glycans and carbohydrates are known for the large number of isomeric features associated with their structures, making analysis challenging compared with other biomolecules. Mass spectrometry has become the primary method of structural characterization for carbohydrates, glycans, and glycoconjugates. Metal adduction is especially important for the mass spectrometric analysis of carbohydrates and glycans. Metal‐ion adduction to carbohydrates and glycoconjugates affects ion formation and the three‐dimensional, gas‐phase structures. Herein, we discuss how metal‐ion adduction impacts ionization, ion mobility, ion activation and dissociation, and hydrogen/deuterium exchange for carbohydrates and glycoconjugates. We also compare the use of different metals for these various techniques and highlight the value in using metals as charge carriers for these analyses. Finally, we provide recommendations for selecting a metal for analysis of carbohydrate adducts and describe areas for continued research.
... The most significant advantage of MS is the ability to obtain detailed structural information of glycans, which makes MS the best tool for analyzing glycosylations. Matrix-assisted laser desorption/ionization MS (MALDI-MS) and electrospray ionization MS (ESI-MS) are two commonly used MS approaches (130,131). MALDI is often combined with time-of-flight (TOF) MS, which has a theoretically infinite m/z range and fast scan speed, enabling extremely low detection lines. ESI is a form of soft ionization protonation that progressively desolvates the sample and forms analyte ions at lower temperatures. ...
... ESI is a form of soft ionization protonation that progressively desolvates the sample and forms analyte ions at lower temperatures. A major advantage of ESI is that it can be easily combined with high performance liquid chromatography (HPLC) to pre-separate complex mixtures of glycans (130,131). Generally, two types of protein glycosylation MS analyses are used, including top-down and bottom-up (80) ( Figure 5). Top-down glycoprotein analysis is the direct assessment of PTMs of the protein backbone without any digestion, which A B C FIGURE 4 Lectin microarray analysis of protein glycosylation. ...
Glycosylation is one of the most important post-translational modifications (PTMs) in a protein, and is the most abundant and diverse biopolymer in nature. Glycans are involved in multiple biological processes of cancer initiation and progression, including cell-cell interactions, cell-extracellular matrix interactions, tumor invasion and metastasis, tumor angiogenesis, and immune regulation. As an important biomarker, tumor-associated glycosylation changes have been extensively studied. This article reviews recent advances in glycosylation-based biomarker research, which is useful for cancer diagnosis and prognostic assessment. Truncated O-glycans, sialylation, fucosylation, and complex branched structures have been found to be the most common structural patterns in malignant tumors. In recent years, immunochemical methods, lectin recognition-based methods, mass spectrometry (MS)-related methods, and fluorescence imaging-based in situ methods have greatly promoted the discovery and application potentials of glycomic and glycoprotein biomarkers in various cancers. In particular, MS-based proteomics has significantly facilitated the comprehensive research of extracellular glycoproteins, increasing our understanding of their critical roles in regulating cellular activities. Predictive, preventive and personalized medicine (PPPM; 3P medicine) is an effective approach of early prediction, prevention and personalized treatment for different patients, and it is known as the new direction of medical development in the 21st century and represents the ultimate goal and highest stage of medical development. Glycosylation has been revealed to have new diagnostic, prognostic, and even therapeutic potentials. The purpose of glycosylation analysis and utilization of biology is to make a fundamental change in health care and medical practice, so as to lead medical research and practice into a new era of 3P medicine.
