Quantitative clinical glycomics strategies: A guide for selecting the best analysis approach

Abstract

Glycans introduce complexity to the proteins to which they are attached. These modifications vary during the progression of many diseases; thus, they serve as potential biomarkers for disease diagnosis and prognosis. The immense structural diversity of glycans makes glycosylation analysis and quantitation difficult. Fortunately, recent advances in analytical techniques provide the opportunity to quantify even lowabundant glycopeptides and glycans derived from complex biological mixtures, allowing for the identification of glycosylation differences between healthy samples and those derived from disease states. Understanding the strengths and weaknesses of different quantitative glycomics analysis methods is important for selecting the best strategy to analyze glycosylation changes in any given set of clinical samples. To provide guidance towards selecting the proper approach, we discuss four widely used quantitative glycomics analysis platforms, including fluorescence-based analysis of released N-linked glycans and three different varieties of MS-based analysis: liquid chromatography (LC)-mass spectrometry (MS) analysis of glycopeptides, matrixassisted laser desorption ionization-time of flight MS, and LC-ESI-MS analysis of released N-linked glycans. These methods' strengths and weaknesses are compared, particularly associated with the figures of merit that are important for clinical biomarker studies, including: the initial sample requirements, the methods' throughput, sample preparation time, the number of species identified, the methods' utility for isomer separation and structural characterization, method-related challenges associated with quantitation, repeatability, the expertise required, and the cost for each analysis. This review, therefore, provides unique guidance to researchers who endeavor to undertake a clinical glycomics analysis by offering insights on the available analysis technologies.

Analysis of Glycosylation and Disulfide Bonding of Wild-Type SARS-CoV-2 Spike Glycoprotein

“…These percentages are obtained by tallying the relative proportion of high-mannose or processed glycoforms, where each glycoform is weighted equally. The percentages are not meant to correspond to the absolute glycan abundance of high-mannose or processed glycoforms, a quantity that is not precisely knowable, since different glyopeptide glycoforms have different ionization efficiencies ( 108 ).…”

The opportunity cost of automated glycopeptide analysis: case study profiling the SARS-CoV-2 S glycoprotein

“…have long known that a glycopeptide interacts with the stationary phase of a C18 column through its peptide portion. Consequently, all the glycoforms from the same peptide will have about the same retention time on a reverse-phase column, with small shifts potentially occurring due to differences in the number of sialic acids [12,33,34]. We capitalized on this information to identify incorrectly assigned glycopeptides in the Byonic-assigned data.…”

“…While the analysis of glycosylation on proteins can be conducted by either releasing the glycans or analyzing glycopeptides [12][13][14], the glycopeptide-based approach is most desirable for multiply glycosylated viral proteins, since analyzing glycopeptides affords the opportunity to identify the glycosylation pattern at individual sites. In both the established field of research in support of HIV-1 vaccine development, where the Env glycoprotein is the target immunogen [3,4,8,9,11,15], and the emerging field of SARS-CoV-2 studies, where the spike (S) glycoprotein is the primary immunogen of interest [16][17][18][19], researchers predominantly use the glycopeptide-based analysis approach.…”

The Hitchhiker's guide to glycoproteomics

“…Understanding the molecular basis of how glycans are involved in health and disease requires technologies to precisely determine both the glycan structures (glycomics), and characterise their location and structure at discrete sites on glycoproteins (glycoproteomics) expressed by a cell or in an entire organ, body fluid, tissue or organism of interest. While the literature harbours many excellent technical reviews covering specific aspects of glycomics (such as [24][25][26][27][28][29][30][31][32][33][34]) and glycoproteomics (examples include [35][36][37][38][39][40][41][42][43][44]) technologies and methodologies, there is a gap in the literature surveying the methods and practical issues of modern glycoproteomics relevant to beginners in the field. This mini-review intends to provide a concise introduction to the current strategies available to generate glycoproteomics data and to provide some guidance for designing tailored glycoproteomics experiments.…”