An official website of the United States government
Chromatography is a robust and reliable separation method that can use various stationary phases to separate complex mixtures commonly seen in metabolomics. This review examines the types of chromatography and stationary phases that have been used in targeted or untargeted metabolomics with methods such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. General considerations for sample pretreatment and separations in metabolomics are considered, along with the various supports and separation formats for chromatography that have been used in such work. The types of liquid chromatography (LC) that have been most extensively used in metabolomics will be examined, such as reversed-phase liquid chromatography and hydrophilic liquid interaction chromatography. In addition, other forms of LC that have been used in more limited applications for metabolomics (e.g., ion-exchange, size-exclusion, and affinity methods) will be discussed to illustrate how these techniques may be utilized for new and future research in this field. Multidimensional LC methods are also discussed, as well as the use of gas chromatography and supercritical fluid chromatography in metabolomics. In addition, the roles of chromatography in NMR- vs. MS-based metabolomics are considered. Applications are given within the field of metabolomics for each type of chromatography, along with potential advantages or limitations of these separation methods.
more »
« less
This content will become publicly available on March 1, 2026
Analysis of solution-phase biomolecular interactions by liquid chromatography: General strategies and recent developments
The analysis of biomolecular interactions is important in characterizing and understanding many fundamental processes that occur in the body and biological systems. A variety of methods are available for studying the extent and rate of binding of these interactions. Some of these techniques are homogeneous methods, with all interacting components being present in the solution-phase, while others are heterogeneous, such as involving both solution-phase and solid-phase components. LC and HPLC have often been used to study biomolecular processes. Although these chromatographic methods make use of both a liquid phase (i.e., the mobile phase and applied samples) and a solid phase (the stationary phase and support), they can be used to study solution-phase interactions. This review examines several strategies that have been developed and employed to use LC and HPLC for this purpose. These strategies include the Hummel-Dreyer method, solution-phase frontal analysis, and the use of physical entrapment for a soluble component of a biomolecular interaction. Other strategies that are discussed are those in which the stationary phase of the column is used as a secondary component or capture agent when studying a solution-phase interaction, as occurs in normal-role affinity chromatography and ultrafast affinity extraction. The general principles for each of these strategies will be considered, along with their advantages, potential limitations, and applications.
more »
« less
- PAR ID:
- 10561559
- Publisher / Repository:
- Elsevier
- Date Published:
- Journal Name:
- Journal of Pharmaceutical and Biomedical Analysis
- Volume:
- 255
- Issue:
- C
- ISSN:
- 0731-7085
- Page Range / eLocation ID:
- 116632
- Subject(s) / Keyword(s):
- Biomolecular interactions Hummel-Dreyer method frontal analysis entrapment normal-role affinity chromatography ultrafast affinity extraction
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
null (Ed.)Adsorption of biomolecules onto material surfaces involves a potentially complex mechanism where molecular species interact to varying degrees with a heterogeneous material surface. Surface adsorption studies by atomic force microscopy, sum frequency generation spectroscopy, and solid-state NMR detect the structures and interactions of biomolecular species that are bound to material surfaces, which, in the absence of a solid–liquid interface, do not exchange rapidly between surface-bound forms and free molecular species in bulk solution. Solution NMR has the potential to complement these techniques by detecting and studying transiently bound biomolecules at the liquid–solid interface. Herein, we show that dark-state exchange saturation transfer (DEST) NMR experiments on gel-stabilized TiO2 nanoparticle (NP) samples detect several forms of biomolecular adsorption onto titanium(IV) oxide surfaces. Specifically, we use the DEST approach to study the interaction of amino acids arginine (Arg), lysine (Lys), leucine (Leu), alanine (Ala), and aspartic acid (Asp) with TiO2 rutile NP surfaces. Whereas Leu, Ala, and Asp display only a single weakly interacting form in the presence of TiO2 NPs, Arg and Lys displayed at least two distinct bound forms: a species that is surface bound and retains a degree of reorientational motion and a second more tightly bound form characterized by broadened DEST profiles upon the addition of TiO2 NPs. Molecular dynamics simulations indicate different surface bound states for both Lys and Arg depending on the degree of TiO2 surface hydroxylation but only a single bound state for Asp regardless of the degree of surface hydroxylation, in agreement with results obtained from the analysis of DEST profiles.more » « less
-
Grinberg, Nelu; Carr, Peter W. (Ed.)Affinity chromatography is a technique that uses a stationary phase based on the supramolecular interactions that occur in biological systems or mimics of these systems. This method has long been a popular tool for the isolation, measurement, and characterization of specific targets in complex samples. This review discusses the basic concepts of this method and examines recent developments in affinity chromatography and related supramolecular separation methods. Topics that are examined include advances that have occurred in the types of supports, approaches to immobilization, and binding agents that are employed in this method. New developments in the applications of affinity chromatography are also summarized, including an overview on the use of this method for biochemical purification, sample preparation or analysis, chiral separations, and biointeraction studies.more » « less
-
Abstract This article describes a protocol for detecting and quantifying RNA phosphorothioate modifications in cellular RNA samples. Starting from solid‐phase synthesis of phosphorothioate RNA dinucleotides, followed by purification with reversed‐phase HPLC, phosphorothioate RNA dinucleotide standards are prepared for UPLC‐MS and LC‐MS/MS methods. RNA samples are extracted from cells using TRIzol reagent, then digested with a nuclease mixture and analyzed by mass spectrometry. UPLC‐MS is employed first to identify RNA phosphorothioate modifications. An optimized LC‐MS/MS method is then employed to quantify the frequency of RNA phosphorothioate modifications in a series of model cells. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis, purification, and characterization of RNA phosphorothioate dinucleotides Basic Protocol 2: Digestion of RNA samples extracted from cells Basic Protocol 3: Detection and quantification of RNA phosphorothioate modifications by mass spectrometrymore » « less
-
Abstract New drug production, from target identification to marketing approval, takes over 12 years and can cost around $2.6 billion. Furthermore, the COVID-19 pandemic has unveiled the urgent need for more powerful computational methods for drug discovery. Here, we review the computational approaches to predicting protein–ligand interactions in the context of drug discovery, focusing on methods using artificial intelligence (AI). We begin with a brief introduction to proteins (targets), ligands (e.g. drugs) and their interactions for nonexperts. Next, we review databases that are commonly used in the domain of protein–ligand interactions. Finally, we survey and analyze the machine learning (ML) approaches implemented to predict protein–ligand binding sites, ligand-binding affinity and binding pose (conformation) including both classical ML algorithms and recent deep learning methods. After exploring the correlation between these three aspects of protein–ligand interaction, it has been proposed that they should be studied in unison. We anticipate that our review will aid exploration and development of more accurate ML-based prediction strategies for studying protein–ligand interactions.more » « less
