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

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 low-abundant 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, matrix-assisted 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.