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1.
Anal Chem ; 95(19): 7475-7486, 2023 05 16.
Article in English | MEDLINE | ID: mdl-37126482

ABSTRACT

Sialic acid isomers attached in either α2,3 or α2,6 linkage to glycan termini confer distinct chemical, biological, and pathological properties, but they cannot be distinguished by mass differences in traditional mass spectrometry experiments. Multiple derivatization strategies have been developed to stabilize and facilitate the analysis of sialic acid isomers and their glycoconjugate carriers by high-performance liquid chromatography, capillary electrophoresis, and mass spectrometry workflows. Herein, a set of novel derivatization schemes are described that result in the introduction of bioorthogonal click chemistry alkyne or azide groups into α2,3- and α2,8-linked sialic acids. These chemical modifications were validated and structurally characterized using model isomeric sialic acid conjugates and model protein carriers. Use of an alkyne-amine, propargylamine, as the second amidation reagent effectively introduces an alkyne functional group into α2,3-linked sialic acid glycoproteins. In tissues, serum, and cultured cells, this allows for the detection and visualization of N-linked glycan sialic acid isomers by imaging mass spectrometry approaches. Formalin-fixed paraffin-embedded prostate cancer tissues and pancreatic cancer cell lines were used to characterize the numbers and distribution of alkyne-modified α2,3-linked sialic acid N-glycans. An azide-amine compound with a poly(ethylene glycol) linker was evaluated for use in histochemical staining. Formalin-fixed pancreatic cancer tissues were amidated with the azide amine, reacted with biotin-alkyne and copper catalyst, and sialic acid isomers detected by streptavidin-peroxidase staining. The direct chemical introduction of bioorthogonal click chemistry reagents into sialic acid-containing glycans and glycoproteins provides a new glycomic tool set to expand approaches for their detection, labeling, visualization, and enrichment.


Subject(s)
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Humans , Sialic Acids/chemistry , Polysaccharides/chemistry , Cell Line, Tumor
2.
Front Pharmacol ; 14: 1337319, 2023.
Article in English | MEDLINE | ID: mdl-38273829

ABSTRACT

Colorectal cancer (CRC) stands as a leading cause of death worldwide, often arising from specific genetic mutations, progressing from pre-cancerous adenomas to adenocarcinomas. Early detection through regular screening can result in a 90% 5-year survival rate for patients. However, unfortunately, only a fraction of CRC cases are identified at pre-invasive stages, allowing progression to occur silently over 10-15 years. The intricate interplay between the immune system and tumor cells within the tumor microenvironment plays a pivotal role in the progression of CRC. Immune cell clusters can either inhibit or facilitate tumor initiation, growth, and metastasis. To gain a better understanding of this relationship, we conducted N-glycomic profiling using matrix-assisted laser desorption-ionization mass spectrometry imaging (MALDI-MSI). We detected nearly 100 N-glycan species across all samples, revealing a shift in N-glycome profiles from normal to cancerous tissues, marked by a decrease in high mannose N-glycans. Further analysis of precancerous to invasive carcinomas showed an increase in pauci-mannose biantennary, and tetraantennary N-glycans with disease progression. Moreover, a distinct stratification in the N-glycome profile was observed between non-mucinous and mucinous CRC tissues, driven by pauci-mannose, high mannose, and bisecting N-glycans. Notably, we identified immune clusters of CD20+ B cells and CD3/CD44+ T cells distinctive and predictive with signature profiles of bisecting and branched N-glycans. These spatial N-glycan profiles offer potential biomarkers and therapeutic targets throughout the progression of CRC.

3.
Front Chem ; 9: 734280, 2021.
Article in English | MEDLINE | ID: mdl-34646811

ABSTRACT

Expressed prostatic secretions (EPS), also called post digital rectal exam urines, are proximal fluids of the prostate that are widely used for diagnostic and prognostic assays for prostate cancer. These fluids contain an abundant number of glycoproteins and extracellular vesicles secreted by the prostate gland, and the ability to detect changes in their N-glycans composition as a reflection of disease state represents potential new biomarker candidates. Methods to characterize these N-glycan constituents directly from clinical samples in a timely manner and with minimal sample processing requirements are not currently available. In this report, an approach is described to directly profile the N-glycan constituents of EPS urine samples, prostatic fluids and urine using imaging mass spectrometry for detection. An amine reactive slide is used to immobilize glycoproteins from a few microliters of spotted samples, followed by peptide N-glycosidase digestion. Over 100 N-glycan compositions can be detected with this method, and it works with urine, urine EPS, prostatic fluids, and urine EPS-derived extracellular vesicles. A comparison of the N-glycans detected from the fluids with tissue N-glycans from prostate cancer tissues was done, indicating a subset of N-glycans present in fluids derived from the gland lumens. The developed N-glycan profiling is amenable to analysis of larger clinical cohorts and adaptable to other biofluids.

4.
Methods Mol Biol ; 2271: 303-316, 2021.
Article in English | MEDLINE | ID: mdl-33908016

ABSTRACT

The analysis of N-glycan distributions in formalin-fixed, paraffin-embedded (FFPE) tissues by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is an effective approach for characterization of many disease states. As the workflow has matured and new technology emerged, approaches are needed to more efficiently characterize the isomeric structures of these N-glycans to expand on the specificity of their localization within tissue. Sialic acid chemical derivatization can be used to determine the isomeric linkage (α2,3 or α2,6) of sialic acids attached to N-glycans, while endoglycosidase F3 (Endo F3) can be enzymatically applied to preferentially release α1,6-linked core fucosylated glycans, further describing the linkage of fucose on N-glycans. Here we describe workflows where N-glycans are chemically derivatized to reveal sialic acid isomeric linkages, combined with a dual-enzymatic approach of endoglycosidase F3 and PNGase F to further elucidate fucosylation isomers on the same tissue section.


Subject(s)
Fixatives/chemistry , Formaldehyde/chemistry , Glycoproteins/analysis , Glycoside Hydrolases/metabolism , Paraffin Embedding , Polysaccharides/analysis , Protein Processing, Post-Translational , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tissue Fixation , Animals , Carbohydrate Conformation , Glycosylation , Humans , Isomerism , Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase/metabolism , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/metabolism , Research Design , Substrate Specificity , Workflow
5.
PLoS One ; 15(4): e0231866, 2020.
Article in English | MEDLINE | ID: mdl-32330167

ABSTRACT

We present the geoBoundaries Global Administrative Database (geoBoundaries): an online, open license resource of the geographic boundaries of political administrative divisions (i.e., state, county). Contrasted to other resources geoBoundaries (1) provides detailed information on the legal open license for every boundary in the repository, and (2) focuses on provisioning highly precise boundary data to support accurate, replicable scientific inquiry. Further, all data is released in a structured form, allowing for the integration of geoBoundaries with large-scale computational workflows. Our database has records for every country around the world, with up to 5 levels of administrative hierarchy. The database is accessible at http://www.geoboundaries.org, and a static version is archived on the Harvard Dataverse.


Subject(s)
Databases, Factual , Internationality , Politics , Internet , Quality Control
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