Characterization of transferrin glycoforms in human serum by CE‐UV and CE‐ESI‐MS

Abstract

Human transferrin (Tf) is a model glycoprotein for congenital disorders of glycosylation (CDG) diagnosis. In the last few years, new CE-UV methods for intact Tf glycoforms analysis have been developed using nonvolatile BGEs and organic modifiers. However, the use of these BGEs does not allow the coupling of these procedures with electrospray MS (ESI-MS). In this study, a new CE-UV separation method of Tf glycoforms is developed, using a double-layer stable coating and a volatile BGE based on ammonium acetate. The separation method is optimized using standard Tf and their potential is demonstrated applying the method to the analysis of sera Tf from healthy individuals and CDG patients. The CE-UV separation method has been coupled to ESI-MS detection. Main parameters such as sheath liquid composition are optimized in order to obtain a good sensitivity. The CE-ESI-MS method has also been used in serum samples obtaining the separation of the different proteins present in serum and partial separation of Tf glycoforms. Different mass spectra and deconvoluted molecular masses were obtained for each sialoform, allowing unequivocal glycoform identification.

Capillary electrophoresis of acidic oligosaccharides from human milk

“…The role of CE-MS in biological analysis has been increasing, allowing rapid identification and quantification of target analytes present at trace levels in biological matrix and providing structural characterization of closely related complex biomolecules. For example, CE-MS has been employed to map glycoforms of bacteria [70,71], to separate and characterize glycoform isomers [72,73], and to detect trace levels of carbohydrates [74][75][76]. However, a method for CE-MS of HMOS has yet to be published.…”

Glycosylation analysis of glycoproteins and proteoglycans using capillary electrophoresis‐mass spectrometry strategies

“…In all but one instances reviewed here on-line interfacing to ESI-MS with sheath-liquid interfaces (built as triple-tube sprayers) was employed [51][52][53][54][55][56][57]. As sheath-liquid small alcohols (methanol or 2-propanol) were mixed with aqueous solutions that contained acidic components (specified in Table 1).…”

“…When aiming for the analysis of samples with physiological concentrations of these glycoproteins, further improvements in analyte enrichment are still required to enhance the over-all sensitivities of the methods. Analysis of glycoprotein isoforms in human serum or urine was either aimed for monitoring glycoforms with disease marker function [57] or for monitoring drug application (e.g., erythropoietin (EPO) [56]). …”

“…[57] for samples of protein standard solutions and for protein samples gained from sera of healthy individuals and persons suffering from congenital disorders of glycosylation (CDG) syndrome that is associated with increased concentrations of carbohydrate-deficient variants of transferrin (cf. Fig.…”

A multiway approach for classification and characterization of rabbit liver apothioneins by CE‐ESI‐MS

“…In CE, protein isoforms (e.g. apothioneins of metalloforms, metalloforms or glycoforms) are primarily separated according to their charge-to-mass ratios, and despite its limited selectivity, UV absorbance detection is used extensively for detection [5][6][7]. In recent years, CE coupled online with ICP-MS has proved to be useful for quantitative speciation of several metals in metalloproteins [8].…”