Table I.

Availability and properties of HT glycomics approaches.

Analyte	Pro	Con	Method	Speed	Precision	Structural resolution	Tools for structure and composition assignment	Ref.
Glycan	• Generic approach • Methods available for absolute quantification • High sensitivity • Highest precision and robustness • High level of isomer differentiation	• Sample purity is essential • Information on site- and protein- specificity lost • HT only for  N-glycans	HILIC– UHPLC–FLD	• 1 sample/ 30 min • 96 samples/ 48 h	Average CV of the 10 most abundant peaks over 2 96-well plates: 1.6%	• Differentiation of various constitutional isomers • Sialic acid linkage differentiation for diantennary glycans	• Retention time • (Tandem) MS • Exoglycosidases	(Huffman et al. 2014; Adamczyk et al. 2017;  Reiding et al. 2019)
			CGE–LIF	• 1 sample/ 45 min • 96 samples/ 3 h with 24-capillary CGE	Average CV of the 10 most abundant peaks over 2 96-well plates: 6.9%		• Migration position • Exoglycosidases	(Callewaert et al. 2004;  Ruhaak et al. 2010;  Huffman et al. 2014;  Reiding et al. 2019)
			MALDI–MS	• 1 sample/ 30 s • 96 samples/ 48 min	Average CV of the 10 most abundant peaks over 2 96-well plates: 11.5%	• Sialic acid linkage differentiation for all N-glycans	• (Tandem) MS	(Reiding et al. 2014, 2019; Vreeker et al. 2018)
Glycopeptides (e.g. IgG and IgA glycosylation)	• Protein- and site-specific (no pure proteins required) • High sensitivity • N- and O-glycans	• Optimization of sample preparation often needed • Limited isomer differentiation	LC–MS	• 1 sample/ 13 min • 96 samples/ 24 h	Average CV over all IgG1 glycoforms in the analysis of 20 96-well plates: 8.2%	• No isomer differentiation • Compositional assignment	• (Tandem) MS	(Huffman et al. 2014; Momcilovic et al. 2020)
Intact glycoproteins (e.g. ApoCIII)	• Protein-specific • Often minimal sample preparation • N- and O-glycans • Complete proteoform assessment	• Rather low sensitivity • No isomer differentiation • Low complexity sample needed • No HT applications yet	MALDI–MS	• 1 sample/ min • 96 samples/ 1.6 h	Average CV over all peaks in the analysis of 1 96-well plates: 15%	• No isomer differentiation • Compositional assignment	• (Tandem) MS	(Nicolardi et al. 2013;  Demus et al. 2021)