Glycan Imaging Mass Spectrometry: Progress in Developing Clinical Diagnostic Assays for Tissues, Biofluids, and Cells

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Key points

  • Imaging mass spectrometry (IMS) is a powerful tool to link changes in disease-associated N-linked glycosylation with histopathology features in standard formalin-fixed paraffin-embedded tissue samples.

  • N-glycan IMS has revealed specific carbohydrate structures and motifs associated with a variety of cancers with the potential for development into clinical biomarkers.

  • Advancements in instrumentation and sample preparation can effectively address N-glycan IMS limitations, such as sialic acid

Method overview and applications of N-glycan matrix-assisted laser desorption/ionization imaging mass spectrometry of tissues

MALDI-IMS analysis of proteins in tissues was initially described in 1997 by the Caprioli laboratory,38 and the field has rapidly evolved to facilitate the spatial distribution and identification of all classes of biomolecules using multiple types of mass spectrometers and ionization sources.39, 40, 41 Key to the success of MALDI-IMS in identification of disease-specific biomolecules has been the ability to correlate IMS data with histopathologic classification of tissue subtypes by standard

Limitations and method adaptations for N-glycan matrix-assisted laser desorption/ionization imaging mass spectrometry

There are several limitations of N-glycan IMS that are inherent to the properties of N-glycans, as well as instrumentation and method limitations that are related to the use of MALDI as the ionization source. These limitations include differentiating structural isomers, stability of sialic acid glycans to MALDI ionization, suppression of N-glycan signal because of matrix effects, the detection of glycans with masses greater than m/z = 4000, and decreased sensitivity with increased spatial

Emerging uses of N-glycan matrix-assisted laser desorption/ionization imaging mass spectrometry for cancer tissue biomarkers and diagnostics

In our group, the use of tissue microarrays (TMAs) representing liver, kidney, pancreas, and breast cancers in conjunction with whole tissue slices have allowed larger cohorts of samples to be analyzed.29,36,51,59 The resulting glycan peak lists can be combined with clinical patient data, pathology information, and genetic subtyping information to evaluate detection of glycan changes as potential diagnostic biomarkers. For breast cancer, an initial study evaluated TMAs representing Her2+ and

N-glycan imaging mass spectrometry of clinical biofluids

Blood-based samples are attractive mediums for biomarker detection because of the availability of large clinical cohorts, as well as the cost-effective and minimally invasive manner of collection. Although there have been several MALDI-MS strategies developed for analysis of N-glycan profiles in biofluids, there are a limited number of high-throughput methodologies capable of analyzing the current-day clinical cohorts that commonly contain thousands of samples. One of the largest plasma

N-glycan imaging mass spectrometry of cultured cells

Although liquid chromatography coupled to MS (LC-MS) is the most common technique for analyzing the N-glycan profile of cells,110,111 Angel and colleagues32 used MALDI-IMS to create an alternative method that is rapid and robust. In this novel method, cells are cultured on a conventional cell culture chamber slide array, fixated with neutral buffered formalin, and delipidated with Carnoy solution. Subsequent steps mirror the tissue imaging workflow, except for a few minor alterations. Compared

N-glycan imaging mass spectrometry of immunoarray-captured glycoproteins

Recently developed immunoassays modified for glycosylation analysis of isolated glycoproteins have the potential for robust analysis of large cohorts of clinical samples. Disease-associated alterations in the glycosylation of immunoglobulin G (IgG),112 α1-acid glycoprotein (AGP),113 haptoglobin,114 transferrin,115 and alpha -fetoprotein108 have been shown and could have utility as biomarkers. There have been several methods for high-throughput N-glycan analysis of immunocaptured glycoproteins

Future directions

For each of the indicated applications, multiple steps in the workflow can be optimized to move toward clinical assay implementation. Glycan profiling of biopsy tissues in particular, such as those frequently obtained for liver, prostate, and breast cancer assessments, or kidney for diabetes, could be integrated with current pathology practices. A recent report described a rapid MALDI-IMS analysis workflow for frozen tissues that required only ∼ 5 total minutes of preparation and analysis time

Summary

This article provides an overview of the clinically relevant applications of this rapidly evolving glycan MALDI-IMS methodology and highlights its potential value as a clinical diagnostic tool. Applying N-glycan MALDI-IMS workflows to the study of tissues, cells, and biofluids has expanded the ability to link glycobiology to mechanisms of disease and improved biomarker detection capabilities. The development and integration of technological advancements in mass spectrometers and sample

Acknowledgments

This research was supported in part by the South Carolina Smart State Centers of Economic Excellence (R.R. Drake, A.S. Mehta) and National Institutes of Health grants U01CA242096 (R.R. Drake, A.S. Mehta, P.M. Angel); R41DK124058 (A.S. Mehta), U01CA226052 (A.S. Mehta, R.R. Drake), Biorepository and Tissue Analysis Shared Resource, Hollings Cancer Center, Medical University of South Carolina (P30 CA138313), and T32GM132055 to C.T. McDowell.

Disclosure

R.R. Drake, P.M. Angel, and A.S. Mehta disclose partial

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    C.R K. Blaschke and C.T. McDowell contributed equally to the work.

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