Human serum albumin (HSA) has been used for a long time as a resuscitation fluid in critically ill patients. It is known to exert several important physiological and pharmacological functions. Among them, the antioxidant properties seem to be of paramount importance as they may be implied in the potential beneficial effects that have been observed in the critical care and hepatological settings. The specific antioxidant functions of the protein are closely related to its structure. Indeed, they are due to its multiple ligand-binding capacities and free radical-trapping properties. The HSA molecule can undergo various structural changes modifying its conformation and hence its binding properties and redox state. Such chemical modifications can occur during bioprocesses and storage conditions of the commercial HSA solutions, resulting in heterogeneous solutions for infusion. In this review, we explore the mechanisms that are responsible for the specific antioxidant properties of HSA in its native form, chemically modified forms, and commercial formulations. To conclude, we discuss the implication of this recent literature for future clinical trials using albumin as a drug and for elucidating the effects of HSA infusion in critically ill patients.

We developed a highly sensitive method for profiling of N-glycans released from proteins based on capillary zone electrophoresis coupled to electrospray ionization mass spectrometry (CZE-ESI-MS) and applied the technique to glycan analysis of plasma and blood-derived isolates. The combination of dopant-enriched nitrogen (DEN)-gas introduced into the nanoelectrospray microenvironment with optimized ionization, desolvation, and CZE-MS conditions improved the detection sensitivity up to ∼100-fold, as directly compared to the conventional mode of instrument operation through peak intensity measurements. Analyses without supplemental pressure increased the resolution ∼7-fold in the separation of closely related and isobaric glycans. The developed method was evaluated for qualitative and quantitative glycan profiling of three types of blood isolates: plasma, total serum immunoglobulin G (IgG), and total plasma extracellular vesicles (EVs). The comparative glycan analysis of IgG and EV isolates and total plasma was conducted for the first time and resulted in detection of >200, >400, and >500 N-glycans for injected sample amounts equivalent to <500 nL of blood. Structural CZE-MS2 analysis resulted in the identification of highly diverse glycans, assignment of α-2,6-linked sialic acids, and differentiation of positional isomers. Unmatched depth of N-glycan profiling was achieved compared to previously reported methods for the analysis of minute amounts of similar complexity blood isolates.

Alterations of protein glycosylation can serve as sensitive and specific disease biomarkers. Labeling procedures for improved separation and detectability of oligosaccharides have several drawbacks, including incomplete derivatization, side-products, noticeable desialylation/defucosylation, sample loss, and interference with downstream analyses. Here, we develop a label-free workflow based on high sensitivity capillary zone electrophoresis-mass spectrometry (CZE-MS) for profiling of native underivatized released N-glycans. Our workflow provides a >45-fold increase in signal intensity compared to the conventional CZE-MS approaches used for N-glycan analysis. Qualitative and quantitative N-glycan profiling of purified human serum IgG, bovine serum fetuin, bovine pancreas ribonuclease B, blood-derived extracellular vesicle isolates, and total plasma results in the detection of >250, >400, >150, >310, and >520 N-glycans, respectively, using injected amounts equivalent to <25 ng of model protein and nL-levels of plasma-derived samples. Compared to reported results for biological samples of similar amounts and complexity, the number of identified N-glycans is increased up to ~15-fold, enabling highly sensitive analysis of sample amounts as low as sub-0.2 nL of plasma volume equivalents. Furthermore, highly sialylated N-glycans are identified and structurally characterized, and untreated sialic acid-linkage isomers are resolved in a single CZE-MS analysis.

No abstract

No abstract

No abstract

Antithrombin (AT) is a human plasma glycoprotein that possesses anticoagulant and anti-inflammatory properties. However, the native (active) form of AT is unstable and undergoes conformational changes, leading to latent, cleaved, and heterodimeric forms. The presence of these alternative forms mostly inactive can highly impact the quality and therapeutic activity of pharmaceutical AT preparations. We developed a capillary zone electrophoresis method, based on a neutral polyethylene oxide-coated capillary and a buffer close to physiological conditions, enabling the separation of more than eight forms of AT. Several peaks were identified as native, latent, and heterodimeric forms. The CZE method was reproducible with intraday relative standard deviations less than 0.5 and 2% for migration times and peak areas, respectively. The method was applied to the comparison of AT preparations produced by five competitive pharmaceutical companies, and statistical tests were performed. Important differences in the proportion of each form were highlighted. In particular, one AT preparation was shown to contain a high quantity of heterodimer, and two preparations contained high quantities of latent form. In addition, one AT preparation exhibited additional forms, not yet identified.

No abstract