Characterization of Complex Proteoform Mixtures by Online Nanoflow Ion-Exchange Chromatography-Native Mass Spectrometry.

The characterization of proteins and complexes in biological systems is essential to establish their critical properties and to understand their unique functions in a plethora of bioprocesses. However, it is highly difficult to analyze low levels of intact proteins in their native states (especially those exceeding 30 kDa) with liquid chromatography (LC)-mass spectrometry (MS). Herein, we describe for the first time the use of nanoflow ion-exchange chromatography directly coupled with native MS to resolve mixtures of intact proteins. Reference proteins and protein complexes with molecular weights between 10 and 150 kDa and a model cell lysate were separated using a salt-mediated pH gradient method with volatile additives. The method allowed for low detection limits (0.22 pmol of monoclonal antibodies), while proteins presented nondenatured MS (low number of charges and limited charge state distributions), and the oligomeric state of the complexes analyzed was mostly kept. Excellent chromatographic separations including the resolution of different proteoforms of large proteins (>140 kDa) and a peak capacity of 82 in a 30 min gradient were obtained. The proposed setup and workflows show great potential for analyzing diverse proteoforms in native top-down proteomics, opening unprecedented opportunities for clinical studies and other sample-limited applications.

Characterizing Non-covalent Protein Complexes Using Asymmetrical Flow Field-Flow Fractionation On-Line Coupled to Native Mass Spectrometry.

We report an online analytical platform based on the coupling of asymmetrical flow field-flow fractionation (AF4) and native mass spectrometry (nMS) in parallel with UV-absorbance, multi-angle light scattering (MALS), and differential-refractive-index (UV-MALS-dRI) detectors to elucidate labile higher-order structures (HOS) of protein biotherapeutics. The technical aspects of coupling AF4 with nMS and the UV-MALS-dRI multi-detection system are discussed. The "slot-outlet" technique was used to reduce sample dilution and split the AF4 effluent between the MS and UV-MALS-dRI detectors. The stability, HOS, and dissociation pathways of the tetrameric biotherapeutic enzyme (anticancer agent) l-asparaginase (ASNase) were studied. ASNase is a 140 kDa homo-tetramer, but the presence of intact octamers and degradation products with lower molecular weights was indicated by AF4-MALS/nMS. Exposing ASNase to 10 mM NaOH disturbed the equilibrium between the different non-covalent species and led to HOS dissociation. Correlation of the information obtained by AF4-MALS (liquid phase) and AF4-nMS (gas phase) revealed the formation of monomeric, tetrameric, and pentameric species. High-resolution MS revealed deamidation of the main intact tetramer upon exposure of ASNase to high pH (NaOH and ammonium bicarbonate). The particular information retrieved from ASNase with the developed platform in a single run demonstrates that the newly developed platform can be highly useful for aggregation and stability studies of protein biopharmaceuticals.

Comprehensive two-dimensional gas chromatography- A discussion on recent innovations.

Although comprehensive 2-D GC is an established and often applied analytical method, the field is still highly dynamic thanks to a remarkable number of innovations. In this review, we discuss a number of recent developments in comprehensive 2-D GC technology. A variety of modulation methods are still being actively investigated and many exciting improvements are discussed in this review. We also review interesting developments in detection methods, retention modeling, and data analysis.

Combining Photodegradation in a Liquid-Core-Waveguide Cell with Multiple-Heart-Cut Two-Dimensional Liquid Chromatography.

Photodegradation greatly affects everyday life. It poses challenges when food deteriorates or when objects of cultural heritage fade, but it can also create opportunities applied in advanced oxidation processes in water purification. Studying photodegradation, however, can be difficult because of the time needed for degradation, the inaccessibility of pure compounds, and the need to handle samples manually. A novel light-exposure cell, based on liquid-core-waveguide (LCW) technology, was embedded in a multiple-heart-cut two-dimensional liquid chromatography system by coupling the LCW cell to the multiple-heart-cut valve. The sample was flushed from the heart-cut loops into the cell by an isocratic pump. Samples were then irradiated using different time intervals and subsequently transferred by the same isocratic pump to a second-dimension sample loop. The mixture containing the transformation products was then subjected to the second-dimension separation. In the current setup, about 30-40% of the selected fraction was transferred. Multiple degradation products could be monitored. Degradation was found to be faster when a smaller sample amount was introduced (0.3 µg as compared to 1.5 µg). The system was tested with three applications, that is, fuchsin, a 19th-century synthetic organic colorant, annatto, a lipophilic food dye, and vitamin B complex.

Continuous Flow Biocatalytic Reductive Amination by Co-Entrapping Dehydrogenases with Agarose Gel in a 3D-Printed Mould Reactor.

Herein, we show how the merge of biocatalysis with flow chemistry aided by 3D-printing technologies can facilitate organic synthesis. This concept was exemplified for the reductive amination of benzaldehyde catalysed by co-immobilised amine dehydrogenase and formate dehydrogenase in a continuous flow micro-reactor. For this purpose, we investigated enzyme co-immobilisation by covalent binding, or ion-affinity binding, or entrapment. Entrapment in an agarose hydrogel turned out to be the most promising solution for this biocatalytic reaction. Therefore, we developed a scalable and customisable approach whereby an agarose hydrogel containing the co-entrapped dehydrogenases was cast in a 3D-printed mould. The reactor was applied to the reductive amination of benzaldehyde in continuous flow over 120 h and afforded 47 % analytical yield and a space-time yield of 7.4 g L day-1 using 0.03 mol% biocatalysts loading. This work also exemplifies how rapid prototyping of enzymatic reactions in flow can be achieved through 3D-printing technology.

Fabrication of monolithic frits and columns for chip-based multidimensional separation devices.

In this work, devices for two-dimensional separations are considered. The device contains a flow distributor, a first-dimension channel, and 17 second-dimension outlets. In the design, all connections between the first-dimension channel, the flow distributor, and the second-dimension outlets were tapered, with a minimal diameter of 20 µm. The use of photo-masking is explored for the fabrication of monolithic frits in all tapered connections. Monolithic frits with optimized permeability and length were successfully fabricated in all 33 tapered channels through light-induced polymerization, photo-masking, and selective exposure. The efficacy of the monolithic frits was demonstrated by creating a packed bed of 15-µm particles, confined within the first-dimension channel. The outlet of the first-dimension channel was successfully connected to a mass spectrometer. Effective flow confinement was demonstrated with a reversed-phase separation of a mixture of five standard peptides.

Introduction of Octadecyl-Bonded Porous Particles in 3D-Printed Transparent Housings with Multiple Outlets.

Microfluidic devices for comprehensive three-dimensional spatial liquid chromatography will ultimately require a body of stationary phase with multiple in- and outlets. In the present work, 3D printing with a transparent polymer resin was used to create a simplified device that can be seen as a unit cell for an eventual three-dimensional separation system. Complete packing of the device with 5-µm C18 particles was achieved, with reasonable permeability. The packing process could be elegantly monitored from the pressure profile, which implies that optical transparency may not be required for future devices. The effluent flow was different for each of the four outlets of the device, but all flows were highly repeatable, suggesting that correction for flow-rate variations is possible. The investigation into flow patterns through the device was supported by computational-fluid-dynamics simulations. A proof-of-principle separation of four standard peptides is described, with mass-spectrometric detection for each of the four channels separately. Supplementary Information: The online version contains supplementary material available at 10.1007/s10337-022-04156-w.

Poly(acrylamide-co-N,N'-methylenebisacrylamide) Monoliths for High-Peak-Capacity Hydrophilic-Interaction Chromatography-High-Resolution Mass Spectrometry of Intact Proteins at Low Trifluoroacetic Acid Content.

In this study, we optimized a polymerization mixture to synthesize poly(acrylamide-co-N,N'-methylenebisacrylamide) monolithic stationary phases for hydrophilic-interaction chromatography (HILIC) of intact proteins. Thermal polymerization was performed, and the effects of varying the amount of cross-linker and the porogen composition on the separation performance of the resulting columns were studied. The homogeneity of the structure and the different porosities were examined through scanning electron microscopy (SEM). Further characterization of the monolithic structure revealed a permeable (Kf between 2.5 × 10-15 and 1.40 × 10-13 m2) and polar stationary phase suitable for HILIC. The HILIC separation performance of the different columns was assessed using gradient separation of a sample containing four intact proteins, with the best performing stationary phase exhibiting a peak capacity of 51 in a gradient of 25 min. Polyacrylamide-based materials were compared with a silica-based particulate amide phase (2.7 µm core-shell particles). The monolith has no residual silanol sites and, therefore, fewer sites for ion-exchange interactions with proteins. Thus, it required lower concentrations of ion-pair reagent in HILIC of intact proteins. When using 0.1% of trifluoroacetic acid (TFA), the peak capacities of the two columns were similar (30 and 34 for the monolithic and packed column, respectively). However, when decreasing the concentration of TFA to 0.005%, the monolithic column maintained similar separation performance and selectivity (peak capacity 23), whereas the packed column showed greatly reduced performance (peak capacity 12), lower selectivity, and inability to elute all four reference proteins. Finally, using a mobile phase containing 0.1% formic acid and 0.005% TFA, the HILIC separation on the monolithic column was successfully hyphenated with high-resolution mass spectrometry. Detection sensitivity for protein and glycoproteins was increased and the amount of adducts formed was decreased in comparison with separations performed at 0.1% TFA.

Charge-Based Separation of Acid-Functional Polymers by Non-aqueous Capillary Electrophoresis Employing Deprotonation and Heteroconjugation Approaches.

Water-borne polymers are in ever-increasing demand due to their favorable ecological profile compared to traditional solvent-borne polymer systems. Many water-borne polymer particles are stabilized in aqueous media by the incorporation of acid-functional monomers. Due to the large variety of comonomers applied, these water-borne polymers have various superimposed statistical distributions, which make it challenging to obtain in-depth information regarding incorporation of the acidic monomers. For selective analysis of the incorporated acidic monomers, a charge-based non-aqueous capillary electrophoresis (NACE) separation was developed. Two approaches were developed: (i) deprotonation of the acid functionality with an organically soluble strong base and (ii) heteroconjugation of anions of carboxylic acids with incorporated acid functionality. In both approaches, N-methylpyrrolidone, as a strong solvent for polymers with a favorable relative permittivity for the presence of dissociated ionic species, was used for the separation. It was shown that anions of carboxylic acids specifically associate with the incorporated acid groups in the polymers, resulting in negatively charged complexes that could be separated based on charge-to-size ratio by NACE. Although both approaches give comparable results with respect to acid distribution for acid-functional polymers, the effective mobility of the deprotonated polymers is roughly double that obtained from the heteroconjugation approach. Unlike the heteroconjugation approach, deprotonation conditions were detrimental to the fused-silica capillary, limiting practical use. Polymers with different chemical compositions, molecular weights, and acid contents were subjected to the CE approaches developed. Polymers with varying molecular weight but similar relative acid monomer content were shown to have similar migration times, which confirms that this approach separates polymers based on charge-to-size ratio.

Recent applications of retention modelling in liquid chromatography.

Recent applications of retention modelling in liquid chromatography (2015-2020) are comprehensively reviewed. The fundamentals of the field, which date back much longer, are summarized. Retention modeling is used in retention-mechanism studies, for determining physical parameters, such as lipophilicity, and for various more-practical purposes, including method development and optimization, method transfer, and stationary-phase characterization and comparison. The review focusses on the effects of mobile-phase composition on retention, but other variables and novel models to describe their effects are also considered. The five most-common models are addressed in detail, i.e. the log-linear (linear-solvent-strength) model, the quadratic model, the log-log (adsorption) model, the mixed-mode model, and the Neue-Kuss model. Isocratic and gradient-elution methods are considered for determining model parameters and the evaluation and validation of fitted models is discussed. Strategies in which retention models are applied for developing and optimizing one- and two-dimensional liquid chromatographic separations are discussed. The review culminates in some overall conclusions and several concrete recommendations.

Confinement of Monolithic Stationary Phases in Targeted Regions of 3D-Printed Titanium Devices Using Thermal Polymerization.

In this study, we have prepared thermally initiated polymeric monolithic stationary phases within discrete regions of 3D-printed titanium devices. The devices were created with controllable hot and cold regions. The monolithic stationary phases were first locally created in capillaries inserted into the channels of the titanium devices. The homogeneity of the monolith structure and the interface length were studied by scanning a capacitively coupled conductivity contactless detector (C4D) along the length of the capillary. Homogeneous monolithic structures could be obtained within a titanium device equipped with a hot and cold jacket connected to two water baths. The confinement method was optimized in capillaries. The sharpest interfaces (between monolith and empty channel) were obtained with the hot region maintained at 70 °C and the cold region at 4 or 10 °C, with the latter temperature yielding better repeatability. The optimized conditions were used to create monoliths bound directly to the walls of the titanium channels. The fabricated monoliths were successfully used to separate a mixture of four intact proteins using reversed-phase liquid chromatography. Further chromatographic characterization showed a permeability (Kf) of ∼4 × 10-15 m2 and a total porosity of 60%.

Recent applications of chemometrics in one- and two-dimensional chromatography.

The proliferation of increasingly more sophisticated analytical separation systems, often incorporating increasingly more powerful detection techniques, such as high-resolution mass spectrometry, causes an urgent need for highly efficient data-analysis and optimization strategies. This is especially true for comprehensive two-dimensional chromatography applied to the separation of very complex samples. In this contribution, the requirement for chemometric tools is explained and the latest developments in approaches for (pre-)processing and analyzing data arising from one- and two-dimensional chromatography systems are reviewed. The final part of this review focuses on the application of chemometrics for method development and optimization.

Characterization of Dye Extracts from Historical Cultural-Heritage Objects Using State-of-the-Art Comprehensive Two-Dimensional Liquid Chromatography and Mass Spectrometry with Active Modulation and Optimized Shifting Gradients.

Unbiased characterization of dyes and their degradation products in cultural-heritage objects requires an analytical method which provides universal separation power regardless of dye classes. Dyes are small molecules that vary widely in chemical structure and properties, which renders their characterization by a single method challenging. We have developed a comprehensive two-dimensional liquid chromatography method hyphenated with mass spectrometry and UV-vis detection. We use stationary-phase-assisted modulation to enhance the method in terms of detection limits and solvent compatibility and to reduce the analysis time. The PIOTR program was used to optimize an assembly of shifting second-dimension gradients, which resulted in a high degree of orthogonality (80% in terms of the asterisk concept). The resulting method is universally applicable to all classes of dyes extracted from cultural-heritage objects. Thanks to the high peak capacity and orthogonality, dye components can be separated from chemically similar impurities and degradation products, providing a detailed fingerprint of the dyes mixture in a specific sample. The method was applied to a number of challenging dye extracts from 17th- and 19th-century cultural-heritage objects.

Perspectives on the future of multi-dimensional platforms.

Two-dimensional liquid chromatography (2D-LC) formats have emerged to help address separation problems that are too complex for conventional one-dimensional LC. There are a number of obstacles to the proliferation of 2D-LC that are gradually being removed. Reliable commercial instrumentation has become available and data analysis software is being improved. Detector-sensitivity and phase-system compatibility issues can largely be solved by using active-modulation strategies. The remaining challenge, developing good and fast 2D-LC methods within a reasonable time, may be solved with smart algorithms. The technology platform that has been developed for 2D-LC also creates a number of other possibilities. Between the two separation stages, all kinds of physical (e.g. dissolution) or chemical (e.g. enzymatic or light-induced degradation) processes can be made to take place, allowing a wide variety of experiments to be performed within a single, efficient and automated analysis. All these developments are discussed in this paper and a number of critical issues are identified. A practical example, the characterization of polysorbates by high-resolution comprehensive two-dimensional liquid chromatography in combination with high-resolution mass spectrometry, is described as a culmination of recent developments in 2D-LC and as an illustration of the current state of the art.

Peak-Tracking Algorithm for Use in Automated Interpretive Method-Development Tools in Liquid Chromatography.

A peak-tracking algorithm for chromatograms recorded using liquid chromatography and mass spectrometry was developed. Peaks are tracked across chromatograms using the spectrometric information, the statistical moments of the chromatographic peaks, and the relative retention. The algorithm can be applied to pair chromatographic peaks in two very different chromatograms, obtained for different samples using different methods. A fast version of the algorithm was specifically tailored to process chromatograms obtained during method development or optimization, where a few similar mobile-phase-composition gradients (same eluent components, but different ranges and programming rates) are applied to the same sample for the purpose of obtaining model parameters to describe the retention of sample components. Due to the relative similarity between chromatograms, time-saving preselection protocols can be used to locate a candidate peak in another chromatogram. The algorithm was applied to two different samples featuring isomers. The automatically tracked peaks and the resulting retention parameters generally yielded prediction errors of less than 1%.

Optimizing separations in online comprehensive two-dimensional liquid chromatography.

Online comprehensive two-dimensional liquid chromatography has become an attractive option for the analysis of complex nonvolatile samples found in various fields (e.g. environmental studies, food, life, and polymer sciences). Two-dimensional liquid chromatography complements the highly popular hyphenated systems that combine liquid chromatography with mass spectrometry. Two-dimensional liquid chromatography is also applied to the analysis of samples that are not compatible with mass spectrometry (e.g. high-molecular-weight polymers), providing important information on the distribution of the sample components along chemical dimensions (molecular weight, charge, lipophilicity, stereochemistry, etc.). Also, in comparison with conventional one-dimensional liquid chromatography, two-dimensional liquid chromatography provides a greater separation power (peak capacity). Because of the additional selectivity and higher peak capacity, the combination of two-dimensional liquid chromatography with mass spectrometry allows for simpler mixtures of compounds to be introduced in the ion source at any given time, improving quantitative analysis by reducing matrix effects. In this review, we summarize the rationale and principles of two-dimensional liquid chromatography experiments, describe advantages and disadvantages of combining different selectivities and discuss strategies to improve the quality of two-dimensional liquid chromatography separations.

Nanoparticle Analysis by Online Comprehensive Two-Dimensional Liquid Chromatography combining Hydrodynamic Chromatography and Size-Exclusion Chromatography with Intermediate Sample Transformation.

Polymeric nanoparticles have become indispensable in modern society with a wide array of applications ranging from waterborne coatings to drug-carrier-delivery systems. While a large range of techniques exist to determine a multitude of properties of these particles, relating physicochemical properties of the particle to the chemical structure of the intrinsic polymers is still challenging. A novel, highly orthogonal separation system based on comprehensive two-dimensional liquid chromatography (LC × LC) has been developed. The system combines hydrodynamic chromatography (HDC) in the first-dimension to separate the particles based on their size, with ultrahigh-performance size-exclusion chromatography (SEC) in the second dimension to separate the constituting polymer molecules according to their hydrodynamic radius for each of 80 to 100 separated fractions. A chip-based mixer is incorporated to transform the sample by dissolving the separated nanoparticles from the first-dimension online in tetrahydrofuran. The polymer bands are then focused using stationary-phase-assisted modulation to enhance sensitivity, and the water from the first-dimension eluent is largely eliminated to allow interaction-free SEC. Using the developed system, the combined two-dimensional distribution of the particle-size and the molecular-size of a mixture of various polystyrene (PS) and polyacrylate (PACR) nanoparticles has been obtained within 60 min.

Untargeted Comprehensive Two-Dimensional Liquid Chromatography Coupled with High-Resolution Mass Spectrometry Analysis of Rice Metabolome Using Multivariate Curve Resolution.

In this work, a new strategy for the chemometric analysis of two-dimensional liquid chromatography-high-resolution mass spectrometry (LC × LC-HRMS) data is proposed. This approach consists of a preliminary compression step along the mass spectrometry (MS) spectral dimension based on the selection of the regions of interest (ROI), followed by a further data compression along the chromatographic dimension by wavelet transforms. In a secondary step, the multivariate curve resolution alternating least squares (MCR-ALS) method is applied to previously compressed data sets obtained in the simultaneous analysis of multiple LC × LC-HRMS chromatographic runs from multiple samples. The feasibility of the proposed approach is demonstrated by its application to a large experimental data set obtained in the untargeted LC × LC-HRMS study of the effects of different environmental conditions (watering and harvesting time) on the metabolism of multiple rice samples. An untargeted chromatographic setup coupling two different liquid chromatography (LC) columns [hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC)] together with an HRMS detector was developed and applied to analyze the metabolites extracted from rice samples at the different experimental conditions. In the case of the metabolomics study taken as example in this work, a total number of 154 metabolites from 15 different families were properly resolved after the application of MCR-ALS. A total of 139 of these metabolites could be identified by their HRMS spectra. Statistical analysis of their concentration changes showed that both watering and harvest time experimental factors had significant effects on rice metabolism. The biochemical insight of the effects of watering and harvesting experimental factors on the changes in concentration of these detected metabolites in the investigated rice samples is attempted.

Reducing Dilution and Analysis Time in Online Comprehensive Two-Dimensional Liquid Chromatography by Active Modulation.

Online comprehensive two-dimensional liquid chromatography (LC × LC) offers ways to achieve high-performance separations in terms of peak capacity (exceeding 1000) and additional selectivity to realize applications that cannot be addressed with one-dimensional chromatography (1D-LC). However, the greater resolving power of LC × LC comes at the price of higher dilutions (thus, reduced sensitivity) and, often, long analysis times (>100 min). The need to preserve the separation attained in the first dimension ((1)D) causes greater dilution for LC × LC, in comparison with 1D-LC, and long analysis times to sample the (1)D with an adequate number of second dimension separations. A way to significantly reduce these downsides is to introduce a concentration step between the two chromatographic dimensions. In this work we present a possible active-modulation approach to concentrate the fractions of (1)D effluent. A typical LC × LC system is used with the addition of a dilution flow to decrease the strength of the (1)D effluent and a modulation unit that uses trap columns. The potential of this approach is demonstrated for the separation of tristyrylphenol ethoxylate phosphate surfactants, using a combination of hydrophilic interaction and reversed-phase liquid chromatography. The modified LC × LC system enabled us to halve the analysis time necessary to obtain a similar degree of separation efficiency with respect to UHPLC based LC × LC and of 5 times with respect to HPLC instrumentation (40 compared with 80 and 200 min, respectively), while at the same time reducing dilution (DF of 142, 299, and 1529, respectively) and solvent consumption per analysis (78, 120, and 800 mL, respectively).

Comprehensive Two-Dimensional Liquid Chromatography with Stationary-Phase-Assisted Modulation Coupled to High-Resolution Mass Spectrometry Applied to Proteome Analysis of Saccharomyces cerevisiae.

Stationary-phase-assisted modulation is used to overcome one of the limitations of contemporary comprehensive two-dimensional liquid chromatography, which arises from the combination of a first-dimension column that is typically narrow and long and a second-dimension column that is wide and short. Shallow gradients at low flow rates are applied in the first dimension, whereas fast analyses (at high flow rates) are required in the second dimension. Limitations of this approach include a low sample capacity of the first-dimension column and a high dilution of the sample in the complete system. Moreover, the relatively high flow rates used for the second dimension make direct (splitless) hyphenation to mass spectrometry difficult. In the present study we demonstrate that stationary-phase-assisted modulation can be implemented in an online comprehensive two-dimensional LC (LC × LC) setup to shift this paradigm. The proposed active modulation makes it possible to choose virtually any combination of first- and second-dimension column diameters without loss in system performance. In the current setup, a 0.30 mm internal diameter first-dimension column with a relatively high loadability is coupled to a 0.075 mm internal diameter second-dimension column. This actively modulated system is coupled to a nanoelectrospray high-resolution mass spectrometer and applied for the separation of the tryptic peptides of a six-protein mixture and for the proteome-wide analyses of yeast from Saccharomyces cerevisiae. In the latter application, about 20000 MS/MS spectra are generated within 24 h analysis time, resulting in the identification of 701 proteins.