Analysis of N-glycans and Future Challenges

N-glycan

As one of the most important post-translational modifications of proteins, N-glycans play a key role in biological processes such as cell recognition, immune responses, and disease development. With the rapid development of mass spectrometry technology, N-glycan analysis has evolved from simple composition identification to high-sensitivity and high-resolution fine structure analysis, opening up new avenues for glycobiology research and clinical diagnosis. This review systematically introduces the technical principles and methodological innovations of N-glycan mass spectrometry analysis, application breakthroughs in cancer biomarker discovery, cutting-edge progress in single-cell level analysis, and challenges in large-scale preparation and standardization. From MALDI imaging to ion mobility separation, from stable isotope labeling to artificial intelligence-assisted analysis, N-glycan mass spectrometry technology is driving the transformation of glycobiology from descriptive research to functional exploration at an astonishing speed, providing powerful tools for precision medicine and biomedical research and development.

Technical Principles and Methodological Innovations of N-glycan Mass Spectrometry Analysis

The foundation of mass spectrometry technology constitutes the cornerstone of N-glycan analysis. Compared with traditional biomolecular analysis, N-glycan mass spectrometry analysis faces unique challenges - the isomerism of glycans (the same molecular weight may correspond to multiple structures) and low ionization efficiency. Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) are the two most commonly used ionization technologies, each with its own advantages: MALDI-MS has the characteristics of low sample fragmentation, strong molecular ion peaks, and high sensitivity, making it particularly suitable for analyzing highly polar, thermally unstable, and non-volatile glycan samples; ESI is more easily combined with liquid chromatography to achieve online separation of complex samples. In recent years, the introduction of ion mobility mass spectrometry (IM-MS) has brought revolutionary breakthroughs to glycan analysis. For example, the Agilent 6560 ion mobility LC/Q-TOF combined with high-resolution data processing software (HRdm 2.0) can separate isomers and conformational isomers based on the unique arrival time distribution (ATD) of each glycan, solving the problem that traditional mass spectrometry has difficulty in distinguishing similar structures. This technology not only improves the separation efficiency but also enables the identification of unknown glycans through "fingerprinting" in the absence of standards.

Innovations in sample pretreatment methods have greatly improved analysis sensitivity and throughput. Traditional glycan release usually requires long-term enzymatic digestion (10-18 hours) and harsh conditions, while microwave-assisted rapid enzymatic digestion technology shortens the PNGase F digestion time to 20 minutes while maintaining high efficiency and mild conditions. In terms of derivatization, stable isotope labeling (such as d0/d5-benzoyl chloride) combined with methylation treatment not only solves the problem of in-source/post-source fragmentation of acidic sugars (such as sialylated glycans) in mass spectrometry detection but also realizes the simultaneous quantitative analysis of neutral and acidic sugars, with a dynamic linear range of up to 10 times. To address the challenge of low-abundance glycans in complex biological samples, new enrichment strategies continue to emerge: cotton-based hydrophilic interaction chromatography (cotton-HILIC SPE) can effectively increase the concentration of plasma glycopeptides; the technology based on oxidative release of native glycans (ORNG) uses household bleach to efficiently extract glycans from large amounts of natural materials, combined with cleavable tag chemistry (such as 4-aminobenzoic acid derivative EPAB) and multidimensional chromatography separation, realizing the preparation of gram-scale complex natural N-glycans. These methodological breakthroughs provide diversified solutions for everything from trace clinical samples to large-scale functional research.

Advances in data processing and structure analysis have accelerated glycan functional research. Traditional glycan identification is highly dependent on standards and known spectral libraries, while logical derivatization sequence tandem mass spectrometry (LODES/MS) directly infers the structure of unknown glycans based on the dissociation mechanism of carbohydrates through collision-induced continuous dissociation sequences, without the need for permethylation, reduction, or labeling, and is suitable for all types of N-glycans (high-mannose, hybrid, and complex). In terms of software tools, professional analysis platforms such as Byonic and MSFragger-glyco have realized multi-level quality control from peptide spectrum matching (PSM) to glycan composition level, ensuring that the false matching rate (FDR) is less than 1%. Of particular note is the application of artificial intelligence algorithms to the automated analysis of glycan NMR data. With the accumulation of glycan collections and NMR data, this direction is expected to achieve high-throughput and standardized glycan structure identification.

Technical integration and multi-dimensional analysis represent the future development direction. The nGlycoDIA workflow developed by the Orbitrap Astral mass spectrometer combined with the narrow-window data non-dependent acquisition (nDIA) strategy identifies more than 3,000 unique glycopeptides from glycopeptide-enriched plasma samples within 40 minutes, with a dynamic range covering seven orders of magnitude, and even detects glycosylated cytokines that were previously difficult to observe (such as IL-12A, IL-12B, IL-22, etc.). Spatial-resolution glycan analysis has also made important progress. Imaging technology based on the solariX MALDI FTMS platform has successfully realized the visualization of the distribution of N-glycans in formalin-fixed paraffin-embedded (FFPE) tissues, revealing the complex carbohydrate interactions and unique glycosylation changes in anaplastic thyroid carcinoma tissues. These technical integrations not only improve the depth and breadth of analysis but also provide a new perspective for understanding the spatio-temporal dynamics of glycans in physiological and pathological processes.

Application Breakthroughs of N-glycan Mass Spectrometry in Cancer Biomarker Discovery

The identification of cancer-specific glycosylation changes provides new ideas for early diagnosis. Abnormal glycosylation, as a universal feature of cancer, is manifested in changes in glycan branching patterns, sialylation levels, and fucosylation levels, and these subtle changes can be accurately captured by high-sensitivity mass spectrometry technology. In the study of anaplastic thyroid carcinoma, MALDI-FTMS imaging technology compared the distribution of N-glycans in cancerous and normal tissues and found a series of specific glycoform changes related to malignant transformation, which could be detected when there were no obvious abnormalities in traditional histological examinations, demonstrating the potential of glycan markers in early diagnosis. More notably, a team from Huazhong University of Science and Technology identified seven characteristic N-glycans (H3N4F1, H4N4F1, H5N4F1, H5N5F1, H4N4S1, H5N4F1S1, and H5N5F1S1) through systematic analysis of the serum of ovarian cancer patients. These markers showed significantly different expressions at the early lesion stage, filling the gap in early serum diagnostic markers for ovarian cancer. These findings not only provide new tools for cancer screening but also provide clues for understanding the glycobiological mechanisms of tumor occurrence and development.

High-sensitivity detection methods make the discovery of low-abundance glycan markers possible. The dynamic range of plasma proteins is extremely wide (more than 10 orders of magnitude), and the microheterogeneity of glycoprotein modifications further dilutes the target signal, which poses a severe challenge to analysis technology. The nGlycoDIA strategy effectively reduces non-glycopeptide interference by optimizing the precursor m/z range (955-1655) and isolation window size (3 Th), and can still detect a variety of low-abundance glycoproteins such as factor VIII and epidermal growth factor receptor from unenriched crude plasma samples. The method of stable isotope labeling combined with methylation treatment protects sialic acid from fragmentation, realizes the accurate quantification of acidic sugars, reaches the picomolar level in the detection limit, and has a dynamic range of 10 times, providing a reliable tool for the discovery of subtle glycosylation changes related to cancer. These technological advances have enabled low-abundance glycan markers that were once buried in noise to emerge, greatly expanding the range of biomarkers that can be studied.

The study of the structure-function relationship of glycans has deepened the understanding of cancer mechanisms. Mass spectrometry technology can not only identify the composition of glycans but also analyze fine structural differences, such as sialic acid linkage modes (α2-3 vs α2-6), fucose positions (core or antenna), etc. These structural details are often related to specific biological functions. For example, the binding specificity of influenza viruses to human cell receptors depends on the sialic acid linkage mode (human influenza viruses prefer α2-6 linkage, while avian influenza viruses tend to α2-3 linkage). In cancer research, similar structure-function associations have received increasing attention. Ion mobility mass spectrometry analysis shows that certain glycan isomers are specifically enriched in tumor tissues, which may promote tumor progression by affecting immune recognition or growth factor signaling. Mass spectrometry-based glycan structure analysis lays the foundation for the development of anti-cancer strategies targeting specific glycoforms, such as designing antibodies or inhibitors against tumor-related glycans.

Translational medicine applications are moving from the laboratory to the clinic. With the deepening of marker research, glycan analysis has begun to transition from the discovery stage to the validation and application stage. A patented technology demonstrates the application prospect of N-glycan rapid quantitative analysis based on MALDI-MS and stable isotope labeling in tumor screening. This method can complete the whole process from sample processing to data analysis within 2 hours, meeting the requirements of clinical diagnosis for speed and stability. Another study uses mass spectrometry imaging technology to directly locate cancer-related glycan changes on FFPE tissue sections. This technology is compatible with routine pathological examinations and is easy to integrate into existing diagnostic processes. Despite facing challenges in standardization and large-scale validation, glycan mass spectrometry analysis, with its high information content and early detection potential, is gradually becoming an important part of cancer precision medicine, especially showing unique value in cancer types where existing markers perform poorly (such as ovarian cancer).

Cutting-edge Progress in Single-cell and Micro-sample N-glycan Mass Spectrometry Analysis

Technical challenges and breakthroughs in single-cell glycoproteomics analysis. Compared with traditional proteomics, single-cell level N-glycosylation analysis faces three dilemmas: extremely low starting amounts (only picogram-level proteins per single cell), complex microheterogeneity (the same protein may carry multiple glycoforms), and the lack of sensitive and efficient analysis methods. In response to these challenges, the team from the Academy of Military Medical Sciences has developed an innovative "signal carrier" strategy - enriching constant samples (40μg peptides) by hydrophilic interaction chromatography (HILIC) to obtain N-glycopeptides as carrier channels, mixing them with trace/single-cell samples (labeled with TMT) for mass spectrometry detection, and using the signal intensity of the carrier channels to correct single-cell data, thus overcoming the problem of low efficiency in direct enrichment of ultra-trace samples. This method does not require separate glycopeptide enrichment for single-cell samples but can achieve considerable analysis depth, realizing a key leap from "impossible" to "possible" and opening the door to the study of glycosylation heterogeneity among immune cell subtypes and within tumor cell populations.

Technical process optimization improves the reliability of single-cell analysis. Single-cell N-glycopeptide analysis involves multiple links such as cell separation, protein extraction, enzymatic digestion, isotope labeling, mass spectrometry detection, and data analysis, and each step may introduce variation or loss. The optimized experimental protocol shows that 10 minutes of heat shock at 95°C combined with sonication can achieve efficient single-cell lysis; 8 hours of tryptic digestion at 37°C ensures the full release of peptides; TMT10-plex labeling followed by room-temperature incubation and hydroxylamine quenching ensures labeling efficiency and reproducibility. In terms of mass spectrometry detection, the configuration of a nano-liquid phase system coupled with a FAIMS Pro interface significantly improves the separation ability, while the open-source software MSFragger-glyco realizes multi-level quality control (FDR<1%) from PSM, peptides to proteins and glycan composition levels. These detailed optimizations together constitute a reliable single-cell glycoproteomics analysis system, enabling researchers to capture rare variations and subpopulation characteristics that are averaged out by traditional population measurements.

Biological discoveries and application prospects. Single-cell resolution reveals the glycosylation heterogeneity that was previously covered up, providing a new perspective for understanding intercellular communication and functional regulation. In immune research, different subtypes of T cells, B cells, and dendritic cells may participate in immune recognition and signal transduction through the specific glycoforms of surface glycoproteins; in the tumor microenvironment, the specific glycosylation patterns of circulating tumor cells and tumor stem cells may be related to their metastatic potential and treatment resistance. In addition, the integrated analysis of single-cell glycoproteomics with transcriptomics and epigenomics is expected to reveal the molecular mechanisms and network relationships of glycosylation regulation. With the further popularization and standardization of the technology, single-cell N-glycan analysis will become a powerful tool for immune therapy response prediction, tumor evolution tracking, and stem cell differentiation research, promoting the deeper development of precision medicine.

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Technical limitations and future directions. Current single-cell N-glycan mass spectrometry analysis still faces several bottlenecks: limited throughput (up to 10 single cells per run), insufficient coverage (only high-abundance glycoproteins can be detected), and complex data analysis (professional bioinformatics support is required). Future breakthroughs may come from several directions: new labeling strategies (such as isotope tags that improve multiplexing capabilities), more sensitive mass spectrometry platforms (such as Orbitrap Astral), and the development of intelligent data analysis tools. Of particular note is the multi-dimensional integration of single-cell glycoproteomics with other omics (such as metabolomics and lipidomics), which is expected to build a more comprehensive cell function map and add a glycobiology perspective to systems biology research.

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