72 research outputs found
Evaluation of capillary electrophoresis-mass spectrometry for the analysis of the conformational heterogeneity of intact proteins using beta2-microglobulin as model compound.
No full textIn this work we explored the feasibility of different CE-ESI-MS set-ups for the analysis of conformational
states of an intact protein. By using the same background electrolyte at quasi physiological conditions
(50 mM ammonium bicarbonate, pH 7.4) a sequential optimization was carried out, initially by evaluating
a sheath-liquid interface with both a single quadrupole (SQ) and a time-of-flight (TOF) mass
spectrometer; then a sheathless interface coupled with high-resolution QTOF MS was considered. Beta2-
microglobulin has been taken as a model, as it is an amyloidogenic protein and its conformational
changes are strictly connected to the onset of a disease. The separation of two conformers at dynamic
equilibrium is achieved all the way down to the MS detection. Notably, the equilibrium ratio of the
protein conformers is maintained in the electrospray source after CE separation. Strengths and weaknesses
of each optimized set-up are emphasized and their feasibility in unfolding studies is evaluated. In
particular, ESI-TOF MS can assign protein forms that differ by 1 Da only and sheathless interfacing is best
suited to preserve protein structure integrity. This demonstrates the CE-ESI-MS performance in terms of
separation, detection and characterization of conformational species that co-populate a protein solution
Online Affinity Assessment and Immunoaffinity Sample Pretreatment in Capillary Electrophoresis-Mass Spectrometry
No full textCapillary electrophoresis (CE) has emerged as a very useful technique for the analysis of a variety of components ranging from small ions to large biomolecules. CE provides efficient separations and short analysis times, and allows compound analysis under near-physiological conditions. In the early 1990s of the last century the capability of CE to assess biomolecular affinity interactions was demonstrated and coined affinity capillary electrophoresis (ACE). In the same time, the use of affinity materials for selective online extraction of compounds before CE analysis was established. This immunoaffinity (IA) CE approach aims for highly specific isolation and preconcentration of target analytes from a biological matrix. Currently, both ACE and IA-CE have developed into proven analytical approaches. Over the last two decades, CE coupled to mass spectrometry (MS) has been demonstrated to be a powerful hyphenated technique, combining the high separation efficiency of CE with the selectivity of MS. MS has also been introduced as a detection technique for both ACE and IA-CE. This chapter provides an overview of the developments and applications in ACE-MS and IA-CE-MS. First, the basic aspects of CE, ACE, and IA-CE are introduced. Subsequently, the hyphenation of CE and MS detection is treated, specifically highlighting aspects that are important for affinity determinations. The setup and performance of reported ACE-MS and IA-CE-MS methods are treated systematically and orderly tables summarizing practical aspects of each method are provided. The application of ACE-MS and IA-CE-MS is outlined treating typical examples, such as peptide library screening, study of protein-ligand interactions, and bioanalysis of peptides and proteins
High-resolution glycoform profiling of intact therapeutic proteins by hydrophilic interaction chromatography-mass spectrometry
No full textGlycosylation is considered a critical quality attribute of therapeutic proteins. Protein heterogeneity introduced by glycosylation includes differences in the nature, number and position of the glycans. Whereas analysis of released glycans and glycopeptides provides information about the composition and/or position of the glycan, intact glycoprotein analysis allows assignment of individual proteoforms and co-occurring modifications. Yet, resolving protein glycoforms at the intact level is challenging. We have explored the capacity of hydrophilic liquid chromatography-mass spectrometry (HILIC-MS) for assessing glycosylation patterns of intact pharmaceutical proteins by analyzing the complex glycoproteins interferon-beta-1a (rhIFN-β − 1a) and recombinant human erythropoietin (rhEPO). Efficient glycoform separation was achieved using a superficially-porous amide HILIC stationary phase and trifluoroacetic acid (TFA) as eluent additive. In-source collision-induced dissociation proved to be very useful to minimize protein-signal suppression effects by TFA. Direct injection of therapeutic proteins in aqueous formulation was possible without causing extra band dispersion, provided that the sample injection volume was not larger than 2 μL. HILIC-MS of rhIFN-β − 1a and rhEPO allowed the assignment of, respectively, 15 and 51 glycoform compositions, next to a variety of posttranslational modifications, such as succinimide, oxidation and N-terminal methionine-loss products. MS-based assignments showed that neutral glycan units significantly contributed to glycoform separation, whereas terminal sialic acids only had a marginal effect on HILIC retention. Comparisons of HILIC-MS with the selectivity provided by capillary electrophoresis-MS for the same glycoproteins, revealed a remarkable complementarity of the techniques. Finally it was demonstrated that by replacing TFA for difluoroacetic acid, peak resolution somewhat decreased, but rhEPO glycoforms with relative abundances below 1% could be detected by HILIC-MS, increasing the overall rhEPO glycoform coverage to 72
Online Affinity Assessment and Immunoaffinity Sample Pretreatment in Capillary Electrophoresis-Mass Spectrometry
No full textHigher-order-structure analysis of proteins by native size-based separations coupled to optical and mass-spectrometric detectors
No full textContinuous developments focussed on ever larger and more-complex molecules, including their supramolecular assemblies, have opened new frontiers and opportunities in many domains of chemistry and, especially, in life sciences, medicine, and biotechnology. Biopharmaceuticals and biotechnological proteins are among the most sophisticated and elegant products of modern science. These highly complex structures possess advanced properties and improved functions, which are directly related to their higher-order structure (HOS) and chemical composition. To develop such products and to ensure their efficacy and safety, suitable analytical methods are crucial. Preservation of the structural integrity of the HOS and the functional native form during analysis is an absolute prerequisite to draw meaningful and reliable conclusions, especially in the characterization of labile macromolecules. In this project the objective was to establish novel analytical platforms by combining advanced separation techniques with state-of-the-art mass spectrometry. The developed platforms provide detailed information on the size, chemical composition and conformation of labile (bio)macromolecules and their HOS in a single analysis. We have performed extensive investigations into the suitability of the size-based separation techniques, mainly size-exclusion chromatography and asymmetrical flow field-flow fractionation, for the non-destructive characterization of proteins under (near-) native conditions. Emphasis has been on optimization of the separation and interfacing with several detectors that provide complementary structural and chemical information. The latter include liquid-phase characterization by multi-angle light scattering (MALS), UV absorbance, and refractive-index detection, and gas-phase detection by high-resolution mass spectrometry. Ultimately, these synergistic platforms were proven pivotal to understand possible structural alterations during the analysis and to ensure conditions that can provide a reliable picture of the actual (native) state of proteins and labile protein complexes in solution
Experimental design and measurement uncertainty in ligand binding studies by affinity capillary electrophoresis
No full textIn all life sciences ligand binding assays (LBAs) play a crucial role. Unfortunately these are very error prone. One part of this uncertainty results from the unavoidable random measurement uncertainty, another part can be attributed to the experimental design. To investigate the latter, uncertainty propagation was evaluated as a function of the given experimental design. A design space including the normalized maximum response range (nMRR), the data point position (DPP), the data point range (DPR) and the number of data points (NoDP) was defined. Based on ten measured ms ACE source data sets 20 specific parameter sets were selected by Design of Experiments. Monte Carlo simulations using 100 000 repeats for every parameter set were employed. The resulting measurement uncertainty propagation factors (measurement uncertainty multiplier: MUM) were used to describe the whole design space by polynomial regression. The resulting 5-dimensional response surface was investigated to evaluate the design parameter's influence and to find the minimal uncertainty propagation. It could be shown, that the nMRR is of highest importance, followed by DPP and DPR. Interestingly, the NoDP is less relevant. However, the interactions of the four parameters need to be carefully considered during design optimization. Using at least five data points which cover over 40% of the upper part of the binding hyperbola (DPP > 0.57) the MUM will be minimized (MUM approximately 1.5) when the nMRR is appropriate. It is possible to reduce the measurement uncertainty propagation more than one order of magnitude
Capillary zone electrophoresis-mass spectrometry of intact proteins
No full textCapillary electrophoresis (CE) coupled with mass spectrometry (MS) has proven to be a powerful analytical tool for the characterization of intact proteins. It combines the high separation efficiency, short analysis time, and versatility of CE with the mass selectivity and sensitivity offered by MS detection. This chapter focuses on important practical considerations when applying CE-MS for the analysis of intact proteins. Technological aspects with respect to the use of CE-MS interfaces and application of noncovalent capillary coatings preventing protein adsorption are treated. Critical factors for successful protein analysis are discussed and four typical CE-MS systems are described demonstrating the characterization of different types of intact proteins by CE-MS. These methodologies comprise the use of sheath-liquid and sheathless CE-MS interfaces, and various types of noncovalent capillary coatings allowing efficient and reproducible protein separations. The discussion includes the analysis of lysozyme-drug conjugates and the therapeutic proteins human growth hormone, human interferon-β-1a, and human erythropoietin.</p
CE-MS for Proteomics and Intact Protein Analysis
No full textThis chapter aims to explore various parameters involved in achieving high-end capillary electrophoresis hyphenated to mass spectrometry (CE-MS) analysis of proteins, peptides, and their posttranslational modifications. The structure of the topics discussed in this book chapter is conveniently mapped on the scheme of the CE-MS system itself, starting from sample preconcentration and injection techniques and finishing with mass analyzer considerations. After going through the technical considerations, a variety of relevant applications for this analytical approach are presented, including posttranslational modifications analysis, clinical biomarker discovery, and its growing use in the biotechnological industry.</p
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