Applications of hydrophilic interaction chromatography to amino acids, peptides, and proteins
TL;DR: This review summarizes the recent advances in the analysis of amino acids, peptides, and proteins using hydrophilic interaction chromatography and expects its use for peptide mapping will continue to grow in the future, particularly because this analytical strategy can be combined with reversed-phase liquid chromatography, in a two-dimensional setup, to reach very high resolving power.
Abstract: This review summarizes the recent advances in the analysis of amino acids, peptides, and proteins using hydrophilic interaction chromatography. Various reports demonstrate the successful analysis of amino acids under such conditions. However, a baseline resolution of the 20 natural amino acids has not yet been published and for this reason, there is often a need to use mass spectrometry for detection to further improve selectivity. Hydrophilic interaction chromatography is also recognized as a powerful technique for peptide analysis, and there are a lot of papers showing its applicability for proteomic applications (peptide mapping). It is expected that its use for peptide mapping will continue to grow in the future, particularly because this analytical strategy can be combined with reversed-phase liquid chromatography, in a two-dimensional setup, to reach very high resolving power. Finally, the interest in hydrophilic interaction chromatography for intact proteins analysis is less evident due to possible solubility issues and a lack of suitable hydrophilic interaction chromatography stationary phases. To date, it has been successfully employed only for the characterization of membrane proteins, histones, and the separation of glycosylated isoforms of an intact glycoprotein. From our point of view, the number of hydrophilic interaction chromatography columns compatible with intact proteins (higher upper temperature limit, large pore size, etc.) is still too limited.
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TL;DR: Progress in the understanding of the HILIC technique is covered, principally over the last ten years, including the classification of columns, the factors that control retention and selectivity, and attempts to model the separation process and its kinetics.
164 citations
TL;DR: This review focuses on the recent progress in understanding the retention mechanism, retention models, selectivity, and the kinetic performance of HILIC.
Abstract: With the exponential growth in the application of the HILIC technique, there has been a significant progress in understanding the fundamental aspects of hydrophilic interaction chromatography. The experimental studies tend to be more extensive in terms of the number of stationary phases investigated and the number of probe compounds employed in comparison with the earlier studies; and more theoretical studies in quantitative structure retention relationship (QSRR) and molecular dynamics simulations have also been published and provide molecular-level insights into the retention mechanism. This review focuses on the recent progress in understanding the retention mechanism, retention models, selectivity, and the kinetic performance of HILIC. A better understanding of these fundamental aspects will undoubtedly facilitate more applications of this chromatographic technique in a wider range of fields.
109 citations
TL;DR: This tutorial aims to provide an overview of the most widely adopted methods of performing LC-HRMS-based untargeted metabolomics of biological samples, accompanied by up-to-date literature to guide readers through the preparation and optimization of such a workflow.
77 citations
TL;DR: This work shows that HILIC can be successfully employed for the analysis of therapeutic proteins and mAbs, using mobile phase compositions comprised of between 65 and 80% ACN and 0.1% TFA, showing orthogonality to RPLC and improved separations of glycoforms.
74 citations
References
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TL;DR: Hydrophilic-interaction chromatography fractionations resemble those obtained through partitioning mechanisms, and the chromatography of DNA, in particular, resembles the partitioning observed with aqueous two-phase systems based on polyethylene glycol and dextran solutions.
1,761 citations
TL;DR: The RP-RP system (employing significantly different pH in both RP separation dimensions) had the highest practical peak capacity of 2D-LC systems investigated and was found to provide suitable orthogonality.
Abstract: Two-dimensional liquid chromatography is often used to reduce the proteomic sample complexity prior to tandem mass spectrometry analysis. The 2D-LC performance depends on the peak capacity in both chromatographic dimensions, and separation orthogonality. The peak capacity and selectivity of many LC modes for peptides is not well known, and mathematical characterization for orthogonality is underdeveloped. Consequently, it is difficult to estimate the performance of 2D-LC for peptide separation. The goal of this paper was to investigate a selectivity of common LC modes and to identify the 2D-LC systems with a useful orthogonality. A geometric approach for orthogonality description was developed and applied for estimation of a practical peak 2D-LC capacity. Selected LC modes including various RP, SCX, SEC, and HILIC were combined in 2D-LC setups. SCX-RP, HILIC-RP, and RP-RP 2D systems were found to provide suitable orthogonality. The RP-RP system (employing significantly different pH in both RP separation dimensions) had the highest practical peak capacity of 2D-LC systems investigated.
762 citations
TL;DR: Electrostatic repulsion-hydrophilic interaction chromatography affords convenient separations of highly charged peptides that cannot readily be resolved by other means, including peptides, amino acids, and nucleotides.
Abstract: If an ion-exchange column is eluted with a predominantly organic mobile phase, then solutes can be retained through hydrophilic interaction even if they have the same charge as the stationary phase This combination is termed electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) With mixtures of solutes that differ greatly in charge, repulsion effects can be exploited to selectively antagonize the retention of the solutes that normally would be the best retained This permits the isocratic resolution of mixtures that normally require gradients, including peptides, amino acids, and nucleotides ERLIC affords convenient separations of highly charged peptides that cannot readily be resolved by other means In addition, phosphopeptides can be isolated selectively from a tryptic digest
483 citations
TL;DR: This review discusses the column efficiency of HILIC materials in relation to solute and stationary phase structures, as well as comparisons between particle-packed and monolithic columns.
394 citations
TL;DR: A novel method utilizing “saltless” pH gradient weak cation exchange-hydrophilic interaction liquid chromatography directly coupled to electron transfer dissociation (ETD) mass spectrometry for the automated on-line high throughput characterization of hypermodified combinatorial histone codes is presented.
294 citations