Developments and perspectives in high-throughput protein glycomics: enabling the analysis of thousands of samples

Abstract

Glycans expand the structural complexity of proteins by several orders of magnitude, resulting in a tremendous analytical challenge when including them in biomedical research. Recent glycobiological research is painting a picture in which glycans represent a crucial structural and functional component of the majority of proteins with alternative glycosylation of proteins and lipids being an important regulatory mechanism in many biological and pathological processes. Since inter-individual differences in glycosylation are extensive, large studies are needed to map the structures and understand the role of glycosylation in human (patho)physiology. Driven by these challenges, methods have emerged which can tackle the complexity of glycosylation in thousands of samples, also known as high-throughput glycomics. For facile dissemination and implementation of high-throughput glycomics technology, the sample preparation, analysis as well as data mining, needs to be stable over a long period of time (months/years), amenable to automation, and available to non-specialized laboratories. Current high-throughput glycomics methods mainly focus on protein N-glycosylation and allow to extensively characterize this subset of the human glycome in large numbers of various biological samples. The ultimate goal in high-throughput glycomics is to gain better knowledge and understanding of the complete human glycome, using methods that are easy to adapt and implement in (basic) biomedical research. Aiming to promote wider use and development of high-throughput glycomics, here we present currently available, emerging and prospective methods and some of their applications, revealing a largely unexplored molecular layer of the complexity of life.

LC-MS/MS-PRM Quantification of IgG glycoforms using stable isotope labeled IgG1 Fc glycopeptide standard

“…A 13 C-labeled IgG1 Fc glycopeptide library was prepared by glycosynthase-catalyzed transfer of a corresponding N-glycan oxazoline to the peptide precursors ( Scheme 2 ). Non-fucosylated glycopeptides ( 21, 23–26 ) were transferred with EndoCC-D180H mutant which is more stable than the EndoM-N175Q mutant (another glycosynthase that transglycosylates complex glycans to the non-fucosylated acceptors) and generally give >95% transfer efficiency. Core fucosylated glycopeptides ( 22, 27–30 ) were synthesized with EndoF3-D165A mutant which also achieves >95% glycan transfer in all cases tested.…”

“…M.W. Quantity (mg) Yield Purity (HPLC) 13 C-Fc1P-S2G2 (21) 4366.28 1.49 >90% >90% 13 C-Fc1P-S1G2 (27) 4075.19 1.64 >90% >90% 13 C-Fc1P-G2 (28) 3784.09 0.73 >90% >95% 13 C-Fc1P-G1 (29) 3622.06 1.31 >90% >95% 13 C-Fc1P-G0 (30) 3459.99 1.14 >90% >95% 13 C-Fc1P-G0F (26) 3606.04 1.55 >90% >95% 13 C-Fc1P-S2G2F (22) 4512.33 1.08 >90% >95% 13 C-Fc1P-S1G2F (23) 4221.24 1.75 >90% >95% 13 C-Fc1P-G2F (24) 3930.17 1.72 >90% >95% 13 C-Fc1P-G1F (25) 3768.09 1.49 >90% >95%…”

“…It is therefore of considerable interest to quantify accurately the IgG glycoforms. So far, many analytical methods have been introduced for this purpose [16][17][18][19][20][21][22][23] , including mass spectrometric methods for relative quantification of the IgG glycopeptides 22,23 . In spite of these advances, the quantification of IgG glycoforms and other glycopeptides by targeted mass spectrometric methods has been limited, 21 in contrast to the quantification of metabolites, drugs or proteins, for which well-established analytical approaches with clinical utility have been established [25][26][27] .…”

“…So far, many analytical methods have been introduced for this purpose [16][17][18][19][20][21][22][23] , including mass spectrometric methods for relative quantification of the IgG glycopeptides 22,23 . In spite of these advances, the quantification of IgG glycoforms and other glycopeptides by targeted mass spectrometric methods has been limited, 21 in contrast to the quantification of metabolites, drugs or proteins, for which well-established analytical approaches with clinical utility have been established [25][26][27] . One reason for the limited acceptance of IgG glycoform quantification in clinical samples is the dominant production of less specific glycan fragments (oxonium ions) under CID conditions used typically for the fragmentation of glycopeptides [28][29][30] .…”

Circulating microRNAs as the Potential Diagnostic and Prognostic Biomarkers for Nasopharyngeal Carcinoma

“…Primarily, the concentrations of expressed proteins are dynamic, fluctuating dramatically at times with regard to stress, illness, and/or treatment. Secondly, quantitative discoveries of proteins require advanced high-throughput technologies, which, therefore, lead to many difficulties associated with sample isolation [ 11 , 21 , 22 , 23 ]. Finally, similar to oncoproteins encoded by EBV, there is a lack of further clinical investigations and validations.…”