Multidimensional Separation Research Articles

Multidimensional separation schemes enhance the identification and molecular characterization of low molecular weight proteomes and short open reading frame-encoded peptides in top-down proteomics

Short open reading frame-encoded peptides (SEP) represent a widely undiscovered part of the proteome. The detailed analysis of SEP has, despite inherent limitations such as incomplete sequence coverage, challenges encountered with protein inference, the identification of posttranslational modifications and the assignment of potential N- and C-terminal truncations, predominantly been assessed using bottom-up proteomic workflows. The use of top-down based proteomic workflows is capable of providing an unparalleled level of characterization information, which is of increased importance in the case of alternatively encoded protein products. However, top-down based analysis is not without its own limitations, for which efficient separation prior to MS analysis is a major issue.We established a sample preparation approach for the combined bottom-up and top-down proteomic analysis of SEP. Key improvements were made by the application of solid phase extraction (SPE), which supported enrichment of proteins below ca. 20 kDa, followed by 2D-LC-MS top-down analysis encompassing both HCD and EThcD ion activation. Bottom-up experiments were used to support and confirm top-down data interpretation. This strategy allowed for the top-down characterization of 36 proteoforms mapping to 12 SEP from the archaeon Methanosarcina mazei strain Gö1, with the concurrent detection and identification of several posttranslational modifications in SEP. Biological significanceSmall or short open reading frames (sORF) have been widely neglected in genome research in the past. With their increasing discovery, the question about the presence and molecular function of their translation products, the short open reading frame-encoded peptides (SEP), arises. As these small proteins are usually below the 10 kDa range, the number of peptides identifiable by bottom-up proteomics is limited which hampers both the identification and the recognition of potential posttranslational modifications. The presented top-down approach allowed for the detection of full length SEP, as well as of terminally truncated proteoforms, and further enabled the identification of disulfide bonds in these small proteins. This demonstrates, that this yet widely undiscovered part of the proteome undergoes the same modifications as classical proteins which is an essential step for future understanding of the biological functions of these molecules.

Hydroperoxylated vs Dihydroxylated Lipids: Differentiation of Isomeric Cardiolipin Oxidation Products by Multidimensional Separation Techniques.

In recent years, cardiolipin (CL) oxidation products were recognized as potential markers for mitochondrial dysfunction in conjunction with age related diseases. The analysis of oxidized CL requires powerful analysis techniques due to high structural diversity. In addition, low concentrations of partly labile compounds pose a special challenge, supplemented by the occurrence of isomeric compounds, e.g., hydroperoxylated vs dihydroxylated products. Therefore, we present a hyphenated method based on liquid chromatography coupled to trapped ion mobility spectrometry (TIMS) for separation and tandem mass spectrometry (MS/MS) for structural characterization. This enables comprehensive analysis of an artificially oxidized CL extract of bovine heart. Isomeric oxidation products could be differentiated by mobility-resolved MS/MS fragmentation experiments. Our developed method could help to better understand the physiological role of oxidized CL.

Unravelling the relationship between aroma compounds and consumer acceptance: Coffee as an example.

Sensory analysis is a key method to assess flavor quality and to characterize consumer preference and acceptance, whereas instrumental analysis helps to identify flavor compounds. The combination of sensory analysis and instrumental analysis provides a platform for revealing key flavor compounds associated with consumer liking. This review discusses sensory evaluation, aroma analysis, and separation techniques using coffee as a central theme where possible to explore the aforementioned techniques. Emerging statistical methodologies are discussed along with their role in tying together discrete studies to reveal important flavor compounds that are either positively or negatively associated with consumer liking. Coffee is very widely studied, a fact that may be partially ascribed to its immense popularity in modern society. To this end, more than 100 sensory lexicons have been developed and implemented to describe specific coffee characteristics and around 1,000 volatile compounds have been identified in coffee. As a remarkably complex sample coffee has provided substantial impetus for adoption of new analytical approaches such as multidimensional separation technologies. This review describes common and emerging analytical techniques that have been employed for coffee analysis, with a particular emphasis placed on those associated with determination of volatile compounds. A comprehensive list of volatile compounds reported in coffee from 1959-2014 is included herein.

Multidimensional characterization of separation processes – Part 2: Comparability of separation efficiency

Following the proposal of deriving statistical entropy maps from multidimensional separation curves, an extension is proposed to allow to compare the separation efficiency of various separation processes. This is achieved by integrating the entropy map weighted by the mass distribution of the particles in the feed. This proposal has several advantages: its straightforward extension to multidimensional partition curves, its scalar value (which allows for a natural ranking of processes), its flexibility to adapt to each and every feed, and its non-parametric character. A typical dynamic air classification process of an iron ore is presented as an example.

Review of Three-Dimensional Liquid Chromatography Platforms for Bottom-Up Proteomics.

Proteomics is a large-scale study of proteins, aiming at the description and characterization of all expressed proteins in biological systems. The expressed proteins are typically highly complex and large in abundance range. To fulfill high accuracy and sensitivity of proteome analysis, the hybrid platforms of multidimensional (MD) separations and mass spectrometry have provided the most powerful solution. Multidimensional separations provide enhanced peak capacity and reduce sample complexity, which enables mass spectrometry to analyze more proteins with high sensitivity. Although two-dimensional (2D) separations have been widely used since the early period of proteomics, three-dimensional (3D) separation was barely used by low reproducibility of separation, increased analysis time in mass spectrometry. With developments of novel microscale techniques such as nano-UPLC and improvements of mass spectrometry, the 3D separation becomes a reliable and practical selection. This review summarizes existing offline and online 3D-LC platforms developed for proteomics and their applications. In detail, setups and implementation of those systems as well as their advances are outlined. The performance of those platforms is also discussed and compared with the state-of-the-art 2D-LC. In addition, we provide some perspectives on the future developments and applications of 3D-LC in proteomics.

Generation of a Collision Cross Section Library for Multi-Dimensional Plant Metabolomics Using UHPLC-Trapped Ion Mobility-MS/MS.

The utility of metabolomics is well documented; however, its full scientific promise has not yet been realized due to multiple technical challenges. These grand challenges include accurate chemical identification of all observable metabolites and the limiting depth-of-coverage of current metabolomics methods. Here, we report a combinatorial solution to aid in both grand challenges using UHPLC-trapped ion mobility spectrometry coupled to tandem mass spectrometry (UHPLC-TIMS-TOF-MS). TIMS offers additional depth-of-coverage through increased peak capacities realized with the multi-dimensional UHPLC-TIMS separations. Metabolite identification confidence is simultaneously enhanced by incorporating orthogonal collision cross section (CCS) data matching. To facilitate metabolite identifications, we created a CCS library of 146 plant natural products. This library was generated using TIMS with N2 drift gas to record the TIMSCCSN2 of plant natural products with a high degree of reproducibility; i.e., average RSD = 0.10%. The robustness of TIMSCCSN2 data matching was tested using authentic standards spiked into complex plant extracts, and the precision of CCS measurements were determined to be independent of matrix affects. The utility of the UHPLC-TIMS-TOF-MS/MS in metabolomics was then demonstrated using extracts from the model legume Medicago truncatula and metabolites were confidently identified based on retention time, accurate mass, molecular formula, and CCS.

Chromatographic behavior and characterization of polydisperse surfactants using Ultra-High-Performance Liquid Chromatography hyphenated to High-Resolution Mass Spectrometry

Surfactants are widely used in various petrochemical applications. Thus, it is essential to have highly efficient analytical tools to monitor the different classes of surfactants commonly used. Three among the four known classes of surfactants were studied in the present work: anionic (i.e. alkylbenzene sulfonate, alkyl ether carboxylic acid), nonionic (i.e. alkylcyclohexyl alcohol ethoxylated, alkyl alcohol ethoxylated) and cationic (i.e. alkyl trimethylammonium). Thanks to high-resolution mass spectrometry (HRMS), a useful mass list was created including 119 m/z values (error in mass around 5 ppm). This list was the foundation of a HRMS database, which, for the sake of simplicity, will be further denoted only “database”. To avoid ion competition and streamline attribution of structural formulas for isobar molecules, a suitable chromatographic method was used before MS. The retention behavior of six surfactants (trimethyloctadecylammonium bromide, myristyltrimethylammonium bromide, Brij®C10, Triton X-100 reduced, glycolic acid ethoxylate lauryl ether, 4-dodecylbenzenesulfonic acid) was evaluated under three separation modes: reversed-phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC) and mixed-mode chromatography (combination of anion-exchange and reversed-phase mechanisms). In RPLC mode, six columns were tested including C4, C18, C30, polar-embedded C18, PFP and phenyl chemistries. Two HILIC columns were also tested including bare silica and urea chemistries. An anion-exchange combined with RPLC mechanism was investigated as mixed-mode mechanism. Using ammonium formate at 10 mM as buffer provided the best signal in HRMS. In liquid chromatography, acid conditions (pH 3.5) were preferred, to avoid peak tailing due to residual silanols. The mixed-mode separation mode clearly appears as the best compromise for the characterization of the three surfactants classes. Nevertheless, the orthogonality observed for the separations obtained in HILIC and RPLC modes offers some possibilities for further multidimensional separations.

Salt-free fractionation of complex isomeric mixtures of glycosaminoglycan oligosaccharides compatible with ESI-MS and microarray analysis

Heparin and heparan sulfate (Hp/HS) are linear complex glycosaminoglycans which are involved in diverse biological processes. The structural complexity brings difficulties in separation, making the study of structure-function relationships challenging. Here we present a separation method for Hp/HS oligosaccharide fractionation with cross-compatible solvent and conditions, combining size exclusion chromatography (SEC), ion-pair reversed phase chromatography (IPRP), and hydrophilic interaction chromatography (HILIC) as three orthogonal separation methods that do not require desalting or extensive sample handling. With this method, the final eluent is suitable for structure-function relationship studies, including tandem mass spectrometry and microarray printing. Our data indicate that high resolution is achieved on both IPRP and HILIC for Hp/HS isomers. In addition, the fractions co-eluted in IPRP could be further separated by HILIC, with both separation dimensions capable of resolving some isomeric oligosaccharides. We demonstrate this method using both unpurified reaction products from isomeric synthetic hexasaccharides and an octasaccharide fraction from enoxaparin, identifying isomers resolved by this multi-dimensional separation method. We demonstrate both structural analysis by MS, as well as functional analysis by microarray printing and screening using a prototypical Hp/HS binding protein: basic-fibroblast growth factor (FGF2). Collectively, this method provides a strategy for efficient Hp/HS structure-function characterization.

Modifier Selectivity Effect on Differential Ion Mobility Resolution of Isomeric Drugs and Multidimensional Liquid Chromatography Ion Mobility Analysis.

Cluster formation in the alternating electric field during differential ion mobility is critical for separation selectivity and is governed by two factors. One is the reduced mass, and the other factor is cluster binding energy between an ion and a neutral solvent molecule (modifier). Therefore, separations of isomeric analytes using a modifier can be related to the thermochemistry of the cluster formation, as subtle changes in the molecular structure will affect its charge delocalization and the binding energy with the corresponding modifier will be different. We have examined the relationship between calculated Gibbs free energies of the cluster formation and experimental ion mobility measurements (CoV dispersion plots) considering the most prominent ion-modifier interactions: charge-dipole, dipole-dipole, and charge-quadrupole. To explain selectivity effects due to the modifier, we have selected a series of positional isomers of sulfonamide drugs that were analyzed in positive and negative electrospray and the diastereoisomers ephedrine and pseudoephedrine in positive mode. The following modifiers were investigated: water, linear and branched alcohols, acetonitrile, acetone, toluene, and ethyl acetate. We could demonstrate a dependence of the separation selectivity of the differential mobility on the reduced mass and Gibbs free energy of the cluster formation. These results are supported by thermochemistry calculations (DFT) and interpreted by molecular modeling. Finally, we describe differential mobility spectrometry selectivity tuning for the multidimensional LCxDMS-MS separation of sulfonamide isomers in human plasma.

A Robust and Universal Metaproteomics Workflow for Research Studies and Routine Diagnostics Within 24 h Using Phenol Extraction, FASP Digest, and the MetaProteomeAnalyzer.

The investigation of microbial proteins by mass spectrometry (metaproteomics) is a key technology for simultaneously assessing the taxonomic composition and the functionality of microbial communities in medical, environmental, and biotechnological applications. We present an improved metaproteomics workflow using an updated sample preparation and a new version of the MetaProteomeAnalyzer software for data analysis. High resolution by multidimensional separation (GeLC, MudPIT) was sacrificed to aim at fast analysis of a broad range of different samples in less than 24 h. The improved workflow generated at least two times as many protein identifications than our previous workflow, and a drastic increase of taxonomic and functional annotations. Improvements of all aspects of the workflow, particularly the speed, are first steps toward potential routine clinical diagnostics (i.e., fecal samples) and analysis of technical and environmental samples. The MetaProteomeAnalyzer is provided to the scientific community as a central remote server solution at www.mpa.ovgu.de.

Multiple dimensions for random walks.

Current trends in diffusion NMR and MRI methods development are reviewed. While great efforts are still directed towards further improving the spectral, spatial, and relaxation rate resolution of basic diffusion measurements, recent improvements in magnetic field gradient technology on whole-body scanners have enabled an exciting line of research involving MRI implementations of advanced diffusion NMR methods with motion-encoding gradient waveforms designed for multidimensional separation and correlation of properties like short-time diffusivity, restriction, anisotropy, flow, and exchange, thereby opening up for highly specific characterization of microstructure and heterogeneity in healthy and diseased tissues in a clinical setting.

Research and Application Progress of Micellar Electrokinetic Chromatography in Separation of Proteins

Abstract Micellar electrokinetic chromatography (MEKC) is a hybrid of chromatography and electrophoresis. The separation mechanism is based on the interaction of analytes and pseudo-stationary phase and also the differential mobility in the electric field. Such property of MEKC can be made good use for separation of proteins and peptides, which have high dimensionality. In the past years, either in the form of single dimensional or multi-dimensional separations, MEKC has demonstrated its excellent usability in biomolecule separations. Recently, as the development in CE-MS was revisited, there are also new developments in MEKC-MS hyphenation techniques, which further points to the usefulness of MEKC in analysis of proteins and peptides. In this article, the recent advances of MEKC applied in separation and analysis of proteins were reviewed and the prospect of this technique was anticipated.

Enhancing Biochemical Resolution by Hyperdimensional Imaging Microscopy

Two decades of fast-paced innovation have improved the spatial resolution of fluorescence microscopy to enable molecular resolution with low invasiveness and high specificity. Fluorescence microscopy also enables scientists and clinicians to map and quantitate the physicochemical properties (e.g., analyte concentration, enzymatic activities, and protein-protein interactions) of biological samples. But the biochemical resolving power of fluorescence microscopy is not as well optimized as its spatial resolution. Current techniques typically observe only the individual properties of fluorescence, thus limiting the opportunities for sensing and multiplexing. Here, we demonstrate a new, to our knowledge, imaging paradigm, hyperdimensional imaging microscopy, which quantifies simultaneously and efficiently all the properties of fluorescence emission (excited-state lifetime, polarization, and spectra) in biological samples, transcending existing limitations. Such simultaneous detection of fluorescence features maximizes the biochemical resolving power of fluorescence microscopy, thereby providing the means to enhance sensing capabilities and enable heavily multiplexed assays. Just as multidimensional separation in mass-spectroscopy and multidimensional spectra in NMR have empowered proteomics and structural biology, we envisage that hyperdimensional imaging microscopy spectra of unprecedented dimensionality will catalyze advances in systems biology and medical diagnostics.

Qualitative evaluation of high pH mass spectrometry-compatible reversed phase liquid chromatography for altered selectivity in separations of intact proteins

Intact proteins are increasingly being recognized as potential biomarkers and biotherapeutic agents for cancer and other serious diseases. Low pH reversed phase plays an important role in both single and multidimensional protein separations for resolving complex protein samples prior to mass spectrometric detection. In this work, we evaluated the use of high pH reversed phase liquid chromatography as an alternative chromatographic separation to gain different selectivity while maintaining the high resolving power and MS compatibility of reversed phase separations. The altered selectivity gained by high pH reversed phase liquid chromatography can further help to separate unresolved protein peaks or to increase peak capacity and resolving power of a multidimensional setup for complex biological samples. Hence, we evaluated the use of different MS-friendly buffers, ion pairing reagents, and stationary phases (silica- and polymer-based) at alkaline pH for intact protein separations. The best chromatographic separation, with complementary selectivity to low pH reversed phase, was achieved using triethylammonium bicarbonate at pH 10 and hybrid silica particles.

Silica modified with polymeric amphiphilic nanoparticles as first dimension for multidimensional separation techniques

This paper addresses the evaluation of a new amphiphilic nanoparticle supported on silica and its application as sorbent in on-line solid phase extraction. The investigated sorbent material is a copolymer composed by [2- (Acryloyloxy) ethyl] trimethylammonium chloride (block A) and butyl acrylate (block B) prepared by reversible addition-fragmentation chain transfer. After polymerization the nanoparticles were adsorbed into silica surface by electrostatic attraction providing to the sorbent both, apolar and polar characteristics. After the fundamental studies to understand the main features of the synthesized nanoparticles supported on silica, this sorbent material was packed into an extraction column. This column was connected on line with liquid chromatography-tandem mass spectrometry and utilized for automated online determination of several analytes as Ochratoxin A, azoxystrobin, cyproconazole and difenoconazole, in wine and water samples.

Deeper Chemical Perceptions for Better Traditional Chinese Medicine Standards

Abstract Traditional Chinese medicines (TCMs), a complex system of natural resources with many diverse components, are widely used as approved medicinal agents in China. Quality control of TCMs is a huge challenge for the government and for testing institutes and is associated with numerous scientific issues. Among these considerations include the following questions: How many components are in TCMs? How can the multiple components in TCMs be comprehensively delineated and subsequently characterized? What is the level and range of these (active) metabolites within these multiple-component TCMs, in order to recommend standards? and What are the qualities required for a marker constituent to be selected, and from a practical perspective, how can these components be assessed with low cost and in a short time? All of these factors require significant and deep thinking in order to understand the individualistic chemistry of TCM in order to develop enhanced TCM quality standards for improved and consistent patient care. In this review, the latest exploratory research in TCM chemistry analytical techniques and methods is summarized in order to begin to develop responses to these scientific issues. Advances in these methods have included multidimensional separation for liquid chromatography–high-resolution mass spectrometry (LC–HRMS), smart triggering data-dependent acquisition of LC–HRMS, target analysis with liquid chromatography–mass spectrometry (LC–MS), supercritical fluid chromatography, and data mining of large mass spectrometry (MS) datasets. In addition, two quality strategies have been introduced in order to save reference standards and the analysis time for a TCM quality standard, including the application of the single standard to determine multi-components (SSDMC) and monomethod-heterotrait matrix methods. Finally, a series of future improvements for analytical methods for TCMs are proposed.

Heart-cut nano-LC-CZE-MS for the characterization of proteins on the intact level.

Multidimensional separation techniques play an increasingly important role in separation science, especially for the analysis of complex samples such as proteins. The combination of reversed-phase liquid chromatography in the nanoscale and CZE is especially beneficial due to their nearly orthogonal separation mechanism and well-suited geometries/dimensions. Here, a heart-cut nano-LC-CZE-MS setup was developed utilizing for the first time a mechanical 4-port valve as LC-CE interface. A model protein mixture containing four different protein species was first separated by nano LC followed by a heart-cut transfer of individual LC peaks and subsequent CZE-MS analysis. In the CZE dimension, various glycoforms of one protein species were separated. Improved separation capabilities were achieved compared to the 1D methods, which was exemplarily shown for ribonuclease B and its different glycosylated forms. LODs in the lower μg/mL range were determined, which are considerably lower compared to traditional CZE-MS. In addition, this study represents the first application of an LC-CE-MS system for intact protein analysis. The nano-LC-CZE-MS system is expected to be applicable to various other analytical challenges.

Multi-column technologies in chromatographic analysis

Multi-column chromatographic technologies provide high throughput and high resolution separations through parallel, serial or parallel-serial column combinations. In comparison with classical single-column based chromatography, multi-column chromatography well satisfied the need for separations of large batch samples and highly complex bio-samples, and therefore attracted extensive interests. In this review, we discussed the recent developments in multi-column chromatography and its applications in multidimensional separation, chip chromatography, capillary electrophoresis, stationary-phase screening, and serial column chromatography. We also discussed the limitations and future developments in multi-column chromatography.

Improving deep proteome and PTMome coverage using tandem HILIC-HPRP peptide fractionation strategy.

Despite being orthogonal to reverse-phase separation and valuable for posttranslational modification (PTM) pre-enrichment, hydrophilic interaction liquid chromatography (HILIC) has not been widely adopted for large-scale proteomic applications. Here, we first evaluated the performance of HILIC in comparison with the popular high-pH reverse-phase (HPRP) separation, as the first dimension for tryptic peptide fractionation in a shotgun workflow to characterize the complex 293T cell proteome. The data indicated that the complementary nature of HILIC and HPRP for peptide separation was mainly due to different hydrophobicity preferences. Realizing that uncaptured components from one mode can be resolved in the other mode, we then designed and compared two multidimensional separation schemes using HILIC and HPRP in tandem for peptide prefractionation, in terms of identification efficiency and coverage at peptide, protein, and PTM levels. A total of 22,604 and 23,566 peptides corresponding to 4481 and 4436 proteins from 293T cell lysate were detected using HILIC-HPRP- and HPRP-HILIC-based shotgun proteomics workflow, respectively. In addition, without assistance of enrichment techniques, the tandem fractionation methods aided to identify 46 different PTMs from over 10,000 of spectra using blind modification search algorithm. We concluded that HILIC is a valuable alternative option for peptide prefractionation in a large-scale proteomic study, but can be further augmented with the use of a secondary HPRP separation.

Nanowell-mediated multidimensional separations combining nanoLC with SLIM IM-MS for rapid, high-peak-capacity proteomic analyses.

Mass spectrometry (MS)-based analysis of complex biological samples is essential for biomedical research and clinical diagnostics. The separation prior to MS plays a key role in the overall analysis, with separations having larger peak capacities often leading to more identified species and improved confidence in those identifications. High-resolution ion mobility (IM) separations enabled by Structures for Lossless Ion Manipulation (SLIM) can provide extremely rapid, high-resolution separations and are well suited as a second dimension of separation following nanoscale liquid chromatography (nanoLC). However, existing sample handling approaches for offline coupling of separation modes require microliter-fraction volumes and are thus not well suited for analysis of trace biological samples. We have developed a novel nanowell-mediated fractionation system that enables nanoLC-separated samples to be efficiently preconcentrated and directly infused at nanoelectrospray flow rates for downstream analysis. When coupled with SLIM IM-MS, the platform enables rapid and high-peak-capacity multidimensional separations of small biological samples. In this study, peptides eluting from a 100nL/min nanoLC separation were fractionated into ~ 60 nanowells on a microfluidic glass chip using an in-house-developed robotic system. The dried samples on the chip were individually reconstituted and ionized by nanoelectrospray for SLIM IM-MS analysis. Using model peptides for characterization of the nanowell platform, we found that at least 80% of the peptide components of the fractionated samples were recovered from the nanowells, providing up to ~tenfold preconcentration for SLIM IM-MS analysis. The combined LC-SLIM IM separation peak capacities exceeded 3600 with a measurement throughput that is similar to current one-dimensional (1D) LC-MS proteomic analyses. Graphical abstract A nanowell-mediated multidimensional separation platform that combines nanoLC with SLIM IM-MS enables rapid, high-peak-capacity proteomic analyses.