Hydrophilic interaction chromatography – mass spectrometry for metabolomics and proteomics:state-of-the-art and current trends

Among all the –omics approaches, proteomics and metabolomics have received increased attention over the last decade. Both approaches have reached a certain level of maturity, showing their relevance in numerous clinical applications, including biomarkers discovery, improved diagnosis, staging, and prognosis of diseases, as well as a better knowledge on various (patho-)physiological processes. Analytically, reversed-phase liquid chromatography – mass spectrometry (RPLC-MS) is considered the golden standard in proteomics and metabolomics, due to its ease of use and reproducilibity. However, RPLC-MS alone is not sufficient to resolve the complexity of the proteome, while very polar metabolites are typically poorly retained. In this context, hydrophilic interaction chromatography (HILIC) represents an attractive complementary approach, due to its orthogonal separation mechanism. This review presents an overview of the literature reporting the application of HILIC-MS in metabolomics and proteomics. For metabolomics the focus is on the analysis of bioactive lipids, amino acids, organic acids, and nucleotides/nucleosides, whereas for proteomics the analysis of complex samples and protein post-translational modifications therein using bottom-up, middle up/down proteomics and intact protein analysis is discussed. The review handles the technological aspects related to the use of HILIC-MS in both proteomics and metabolomics, paying attention to stationary phases, mobile phase conditions, injection volume and column temperature. Recent trends and developments in the application of HILIC-MS in proteomics and metabolomics are also presented and discussed, highlighting the advantages the technique can provide in addition or complementary to RPLC-MS, as well as the current limitations and possible solutions

Прогноз глобальних тенденцій релігійних змін у світі ХХІ століття та їхніх імплікацій в українському контексті

Drug purity and affinity are essential attributes during development and production of therapeutic proteins. In this work, capillary electrophoresis (CE) was used to determine both the affinity and composition of the biotechnologically produced "nanobody" EGa1, the binding fragment of a heavy-chain-only antibody. EGa1 is an antagonist of the epidermal growth factor receptor (EGFR), which is overexpressed on the surface of tumor cells. Using a background electrolyte (BGE) of 50mM sodium phosphate (pH 8.0) in combination with a polybrene-poly(vinylsulfonic acid) capillary coating, CE analysis of EGa1 showed the presence of at least three components. Affinity of the EGa1 components towards the extracellular domain of EGFR was assessed by adding different concentrations (0-12nM) of the receptor to the BGE while measuring the effective electrophoretic mobility of the respective EGa1 components. Binding curves obtained by plotting electrophoretic mobility shifts as a function of receptor concentration, yielded dissociation constants (

Microfluidic ion stripper for removal of trifluoroacetic acid from mobile phases used in HILIC-MS of intact proteins

© 2021, The Author(s).Trifluoroacetic acid (TFA) is commonly used as mobile phase additive to improve retention and peak shape characteristics in hydrophilic interaction liquid chromatography (HILIC) of intact proteins. However, when using electrospray ionization-mass spectrometry (ESI-MS) detection, TFA may cause ionization suppression and adduct formation, leading to reduced analyte sensitivity. To address this, we describe a membrane-based microfluidic chip with multiple parallel channels for the selective post-column removal of TFA anions from HILIC. An anion-exchange membrane was used to physically separate the column effluent from a stripper flow solution comprising acetonitrile, formic acid, and propionic acid. The exchange of ions allowed the post-column removal of TFA used during HILIC separation of model proteins. The multichannel design of the device allows the use of flow rates of 0.2 mL/min without the need for a flow splitter, using mobile phases containing 0.1% TFA (13 mM). Separation selectivity and efficiency were maintained (with minor band broadening effects) while increasing the signal intensity and peak areas by improving ionization and reducing TFA adduct formation. Graphical abstract: [Figure not available: see fulltext.

Capillary electrophoresis-mass spectrometry using noncovalently coated capillaries for the analysis of biopharmaceuticals

In this work, the usefulness of capillary electrophoresis–electrospray ionization time-of-flight–mass spectrometry for the analysis of biopharmaceuticals was studied. Noncovalently bound capillary coatings consisting of Polybrene-poly(vinyl sulfonic acid) or Polybrene-dextran sulfate-Polybrene were used to minimize protein and peptide adsorption, and achieve good separation efficiencies. The potential of the capillary electrophoresis-mass spectrometry (CE-MS) system to characterize degradation products was investigated by analyzing samples of the drugs, recombinant human growth hormone (rhGH) and oxytocin, which had been subjected to prolonged storage, heat exposure, and/or different pH values. Modifications could be assigned based on accurate masses as obtained with time-of-flight–mass spectrometry (TOF-MS) and migration times with respect to the parent compound. For heat-exposed rhGH, oxidations, sulfonate formation, and deamidations were observed. Oxytocin showed strong deamidation (up to 40%) upon heat exposure at low pH, whereas at medium and high pH, mainly dimer (>10%) and trisulfide formation (6–7%) occurred. Recombinant human interferon-β-1a (rhIFN-β) was used to evaluate the capability of the CE-MS method to assess glycan heterogeneity of pharmaceutical proteins. Analysis of this N-glycosylated protein revealed a cluster of resolved peaks which appeared to be caused by at least ten glycoforms differing merely in sialic acid and hexose N-acetylhexosamine composition. Based on the relative peak area (assuming an equimolar response per glycoform), a quantitative profile could be derived with the disialytated biantennary glycoform as most abundant (52%). Such a profile may be useful for in-process and quality control of rhIFN-β batches. It is concluded that the separation power provided by combined capillary electrophoresis and TOF-MS allows discrimination of highly related protein species