Serial in-solution digestion protocol for mass spectrometry-based glycomics and proteomics analysis

Sethi, Manveen K.; Downs, Margaret; Zaia, Joseph

doi:10.1039/d0mo00019a

Cited by 22 publications

(18 citation statements)

References 44 publications

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“…3a-b. Previous LC-MS/MS-based studies have shown that other enzymes targeted to GAGs, such as hyaluronidase and heparan lysase, can be used in tandem to chondroitinase ABC to serially access the proteoglycome [5]. Ongoing studies aim to incorporate these enzymes into MALDI-IMS multienzyme workflows.…”

Section: Methods Optimization For Chondroitin Sulfate Imaging Msmentioning

confidence: 99%

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Multiplexed imaging mass spectrometry of the extracellular matrix using serial enzyme digests from formalin-fixed paraffin-embedded tissue sections

et al. 2020

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We report a multiplexed imaging mass spectrometry method which spatially localizes and selectively accesses the extracellular matrix on formalin-fixed paraffin-embedded tissue sections. The extracellular matrix (ECM) consists of (1) fibrous proteins, posttranslationally modified (PTM) via N-and O-linked glycosylation, as well as hydroxylation on prolines and lysines, and (2) glycosaminoglycan-decorated proteoglycans. Accessing all these components poses a unique analytical challenge. Conventional peptide analysis via trypsin inefficiently captures ECM peptides due to their low abundance, intra-and intermolecular crosslinking, and PTMs. In previous studies, we have developed matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) techniques to capture collagen peptides via collagenase type III digestion, both alone and after N-glycan removal via PNGaseF digest. However, in fibrotic tissues, the buildup of ECM components other than collagen-type proteins, including elastin and glycosaminoglycans, limits efficacy of any single enzyme to access the complex ECM. Here, we have developed a novel serial enzyme strategy to define the extracellular matrix, including PTMs, from a single tissue section for MALDI-IMS applications.

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Section: Methods Optimization For Chondroitin Sulfate Imaging Msmentioning

confidence: 99%

“…Enzymatically targeted techniques have been developed recently for multi-omics studies and can analyze tissue glycomics, glycoproteomics, and/or proteomics from a single sample [5][6][7]. These techniques are largely serial LC-MS/MS strategies applied to fresh-frozen tissue samples.…”

Section: Introductionmentioning

confidence: 99%

Multiplexed imaging mass spectrometry of the extracellular matrix using serial enzyme digests from formalin-fixed paraffin-embedded tissue sections

et al. 2020

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“…Multiple protease approaches have already proven their benefit for glycoproteome characterization (86,(462)(463)(464)(465)(466). Even so, these largely rely on canonical proteases like trypsin, chymotrypsin, GluC, and AspN, among others, and some classes of glycosylation, e.g., mucintype O-glycosylation remain impervious to these proteases.…”

Section: Related Developments In Glycoproteomicsmentioning

confidence: 99%

“…Furthermore, glycosites often occur in regions of proteins that can be undersampled with standard proteomic methodologies that rely on trypsin. Multiple protease approaches have already proven their benefit for glycoproteome characterization ( 86 , 460 , 461 , 462 , 463 , 464 ). Even so, these largely rely on canonical proteases like trypsin, chymotrypsin, GluC, and AspN, among others.…”

Section: Related Developments In Glycoproteomicsmentioning

confidence: 99%

A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry–Based Glycoproteomics

Riley

Bertozzi

Pitteri

2021

Molecular & Cellular Proteomics

165

158

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Glycosylation is a prevalent, yet heterogeneous modification with a broad range of implications in molecular biology. This heterogeneity precludes enrichment strategies that can be universally beneficial for all glycan classes. Thus, choice of enrichment strategy has profound implications on experimental outcomes. Here we review common enrichment strategies used in modern mass spectrometry (MS)-based glycoproteomic experiments, including lectins and other affinity chromatographies, hydrophilic interaction chromatography (HILIC) and its derivatives, porous graphitic carbon (PGC), reversible and irreversible chemical coupling strategies, and chemical biology tools that often leverage bioorthogonal handles. Interest in glycoproteomics continues to surge as MS instrumentation and software improve, so this review aims to help equip researchers with necessary information to choose appropriate enrichment strategies that best complement these efforts.

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“…Also, the ability to sequence GAG chains should provide critical insight into structure-function relationships. Progress in mass spectrometry-based glycomics in the past decades has been exciting with the aid of bioinformatics (Ricard-Blum and Lisacek, 2017 ; Sethi et al, 2020 ). Ultimately, glycosamino-glycomics would require the capability of unbiased sequencing of any oligosaccharides derived preferentially from the natural source.…”

mentioning

confidence: 99%

Role of Chondroitin Sulfation Following Spinal Cord Injury

Hussein

Mencio

Katagiri

et al. 2020

Front. Cell. Neurosci.

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Traumatic spinal cord injury produces long-term neurological damage, and presents a significant public health problem with nearly 18,000 new cases per year in the U.S. The injury results in both acute and chronic changes in the spinal cord, ultimately resulting in the production of a glial scar, consisting of multiple cells including fibroblasts, macrophages, microglia, and reactive astrocytes. Within the scar, there is an accumulation of extracellular matrix (ECM) molecules-primarily tenascins and chondroitin sulfate proteoglycans (CSPGs)-which are considered to be inhibitory to axonal regeneration. In this review article, we discuss the role of CSPGs in the injury response, especially how sulfated glycosaminoglycan (GAG) chains act to inhibit plasticity and regeneration. This includes how sulfation of GAG chains influences their biological activity and interactions with potential receptors. Comprehending the role of CSPGs in the inhibitory properties of the glial scar provides critical knowledge in the much-needed production of new therapies.

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Serial in-solution digestion protocol for mass spectrometry-based glycomics and proteomics analysis

Abstract: A high-throughput & efficient protocol for mass spectrometry-based glycomic and proteomic analysis.

Cited by 22 publications

References 44 publications

Multiplexed imaging mass spectrometry of the extracellular matrix using serial enzyme digests from formalin-fixed paraffin-embedded tissue sections

Multiplexed imaging mass spectrometry of the extracellular matrix using serial enzyme digests from formalin-fixed paraffin-embedded tissue sections

A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry–Based Glycoproteomics

Role of Chondroitin Sulfation Following Spinal Cord Injury

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