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Can reversed-phase peptide retention be the same for C8 and C18 columns? or increase for otherwise identical columns with a smaller surface area? Can replacing trifluoroacetic acid (TFA) with formic acid (FA) improve the peak shape? According to our common understanding of peptide chromatography, absolutely not. Surprisingly, a thorough comparison of the peptide separation selectivity of 100 and 120 Å fully porous C18 sorbents to maximize the performance of our in-house proteomics LC-MS/MS setup revealed an unexpectedly higher peptide retentivity for a wider pore packing material, despite it having a smaller surface area. Concurrently, the observed increase in peptide retention─which drives variation in separation selectivity between 100 and 120 Å pore size materials─was more pronounced for smaller peptides. These findings contradict the central dogmas that underlie the development of all peptide RP-HPLC applications: (i) a larger surface area leads to higher retention and (ii) increasing the pore size should benefit the retention of larger analytes. Based on our intriguing findings, we compared reversed-phase high-performance liquid chromatography peptide retention for a total of 20 columns with pore sizes between 60 and 300 Å using FA- and TFA-based eluents. Our results unequivocally attest that the larger size of ion pairs in FA- vs TFA-based eluents leads to the observed impact on selectivity and peptide retention. For FA, peptide retention peaks at 200 Å pore size, compared to between 120 and 200 Å for TFA. However, the decrease in retention for narrow-pore particles is more profound in FA. Our findings suggest that common assumptions about analyte size and accessible surface area should be revisited for ion-pair RP separation of small peptides, typical for proteomic applications that are predominantly applying FA eluents. Hybrid silica-based materials with pore sizes of 130-200 Å should be specifically targeted for bottom-up proteomic applications to obtain both superior peak shape and peptide retentivity. This challenging task of attaining the best RPLC column for proteomics calls for closer collaboration between LC column manufacturers and proteomic LC specialists.

In vitro transcription (IVT) of mRNA is a versatile platform for a broad range of biotechnological applications. Its rapid, scalable, and cost-effective production makes it a compelling choice for the development of mRNA-based cancer therapies and vaccines against infectious diseases. The impurities generated during mRNA production can potentially impact the safety and efficacy of mRNA therapeutics, but their structural complexity has not been investigated in detail yet. This study pioneers a comprehensive profiling of IVT mRNA impurities, integrating current technologies with innovative analytical tools. We have developed highly reproducible, efficient, and stability-indicating ion-pair reversed-phase liquid chromatography and capillary gel electrophoresis methods to determine the purity of mRNA from different suppliers. Furthermore, we introduced the applicability of microcapillary electrophoresis for high-throughput (<1.5 min analysis time per sample) mRNA impurity profiling. Our findings revealed that impurities are mainly attributed to mRNA variants with different poly(A) tail lengths due to aborted additions or partial hydrolysis and the presence of double-stranded mRNA (dsRNA) byproducts, particularly the dsRNA 3'-loop back form. We also implemented mass photometry and native mass spectrometry for the characterization of mRNA and its related product impurities. Mass photometry enabled the determination of the number of nucleotides of different mRNAs with high accuracy as well as the detection of their size variants [i.e., aggregates and partial and/or total absence of the poly(A) tail], thus providing valuable information on mRNA identity and integrity. In addition, native mass spectrometry provided insights into mRNA intact mass, heterogeneity, and important sequence features such as poly(A) tail length and distribution. This study highlights the existing bottlenecks and opportunities for improvement in the analytical characterization of IVT mRNA, thus contributing to the refinement and streamlining of mRNA production, paving the way for continued advancements in biotechnological applications.

Analytical methods for the assessment of drug-delivery systems (DDSs) are commonly suitable for characterizing individual DDS properties, but do not allow determination of several properties simultaneously. A comprehensive online two-dimensional liquid chromatography (LC × LC) system was developed that is aimed to be capable of characterizing both nanoparticle size and encapsulated cargo over the particle size distribution of a DDS by using one integrated method. Polymeric nanoparticles (NPs) with encapsulated hydrophobic dyes were used as model DDSs. Hydrodynamic chromatography (HDC) was used in the first dimension to separate the intact NPs and to determine the particle size distribution. Fractions from the first dimension were taken comprehensively and disassembled online by the addition of an organic solvent, thereby releasing the encapsulated cargo. Reversed-phase liquid chromatography (RPLC) was used as a second dimension to separate the released dyes. Conditions were optimized to ensure the complete disassembly of the NPs and the dissolution of the dyes during the solvent modulation step. Subsequently, stationary-phase-assisted modulation (SPAM) was applied for trapping and preconcentration of the analytes, thereby minimizing the risk of analyte precipitation or breakthrough. The developed HDC × RPLC method allows for the characterization of encapsulated cargo as a function of intact nanoparticle size and shows potential for the analysis of API stability.

Messenger RNA (mRNA) is a new class of therapeutic compounds. The current advances in mRNA technology require the development of efficient analytical methods. In this work, we describe the development of several methods for measurement of mRNA poly(A) tail length and heterogeneity. Poly(A) tail was first cleaved from mRNA with the RNase T1 enzyme. The average length of a liberated poly(A) tail was analyzed with the size exclusion chromatography method. Size heterogeneity of the poly(A) tail was estimated with high-resolution ion-pair reversed phase liquid chromatography (IP RP LC). The IP RP LC method provides resolution of poly(A) tail oligonucleotide variants up to 150 nucleotide long. Both methods use a robust ultraviolet detection suitable for mRNA analysis in quality control laboratories. The results were confirmed by the LC-mass spectrometry (LC MS) analysis of the same mRNA sample. The poly(A) tail length and heterogeneity results were in good agreement.

Peptide separations by reversed-phase liquid chromatography (RPLC) are an integral part of bottom-up proteomics. These separations typically employ C18 columns with water/acetonitrile gradient elution in the presence of formic acid. Despite the widespread use of such workflows, the exact nature of peptide interactions with the stationary and mobile phases is poorly understood. Here, we employ microsecond molecular dynamics (MD) simulations to uncover details of peptide RPLC. We examined two tryptic peptides, a hydrophobic and a hydrophilic species, in a slit pore lined with C18 chains that were grafted onto SiO 2 support. Our simulations explored peptide trapping, followed by desorption and elution. Trapping in an aqueous mobile phase was initiated by C18 contacts with Lys butyl moieties. This was followed by extensive anchoring of nonpolar side chains (Leu/Ile/Val) in the C18 layer. Exposure to water/acetonitrile triggered peptide desorption in a stepwise fashion; charged sites close to the termini were the first to lift off, followed by the other residues. During water/acetonitrile elution, both peptides preferentially resided close to the pore center. The hydrophilic peptide exhibited no contacts with the stationary phase under these conditions. In contrast, the hydrophobic species underwent multiple transient Leu/Ile/Val binding interactions with C18 chains. These nonpolar interactions represent the foundation of differential peptide retention, in agreement with the experimental elution behavior of the two peptides. Extensive peptide/formate ion pairing was observed in water/acetonitrile, particularly at N-terminal sites. Overall, this work uncovers an unprecedented level of RPLC molecular details, paving the way for MD simulations as a future tool for improving retention prediction algorithms and for the design of novel column materials.

Multidimensional liquid chromatography (mD-LC) is becoming a powerful tool for complete characterization of individual peaks and protein variants through separation methods such as nondenaturing ion exchange (IEC) or size-exclusion chromatography coupled to reversed-phase (RP) chromatography. The flexibility of commercially available and customized mD-LC systems is still limited in terms of enzymatic peak processing between chromatographic dimensions. In this regard, only a few column-immobilized proteases are available for detailed peak characterization by mD-LC coupled to mass spectrometry (mD-LC-MS). Here, we present a purpose-built and automated multiple heart-cutting mD-LC design with a novel analytical workflow involving in-loop enzymatic heart-cut digestion between the first-dimensional column and transfer to the second dimension before MS or MS/MS analyses. The setup facilitates the spike-in of any enzyme to multiple heart-cuts for multilevel analysis, for example, for peptide mapping, fragment generation, or deglycosylation, to reduce heterogeneity and provide maximum flexibility in terms of incubation time for optimal peak characterization. We demonstrate the application of IEC coupled to RP-LC-MS and automated in-loop deglycosylation and on-column reduction of an IgG antibody combined with upper hinge region cleavage for Fab generation. We further employ mD-LC-MS and mD-LC-MS/MS to assess post-translational modifications of a bispecific antibody and to support molecule selection by evaluating the best downstream purification strategy. The novel design and automated workflow of the mD-LC system described here offers enhanced flexibility for in-solution processing and real-time monitoring of multiple heart-cuts enabling streamlined characterization of unknown biotherapeutic charge and size variants.

Dolichyl monophosphates (DolPs) are essential lipids in glycosylation pathways that are highly conserved across almost all domains of life. The availability of DolP is critical for all glycosylation processes, as these lipids serve as membrane-anchored building blocks used by various types of glycosyltransferases to generate complex post-translational modifications of proteins and lipids. The analysis of DolP species by reverse-phase liquid chromatography-mass spectrometry (RPLC-MS) remains a challenge due to their very low abundance and wide range of lipophilicities. Until now, a method for the simultaneous qualitative and quantitative assessment of DolP species from biological membranes has been lacking. Here, we describe a novel approach based on simple sample preparation, rapid and efficient trimethylsilyl diazomethane-dependent phosphate methylation, and RPLC-MS analysis for quantification of DolP species with different isoprene chain lengths. We used this workflow to selectively quantify DolP species from lipid extracts derived of Saccharomyces cerevisiae , HeLa, and human skin fibroblasts from steroid 5-α-reductase 3- congenital disorders of glycosylation (SRD5A3-CDG) patients and healthy controls. Integration of this workflow with global lipidomics analyses will be a powerful tool to expand our understanding of the role of DolPs in pathophysiological alterations of metabolic pathways downstream of HMG-CoA reductase, associated with CDGs, hypercholesterolemia, neurodegeneration, and cancer.

Liquid chromatography-mass spectrometry (LC-MS)-based metabolomics usually combines hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP) chromatography to cover a wide range of metabolomes, requiring both significant sample consumption and analysis time for separate workflows. We developed an integrated workflow enabling the coverage of both polar and nonpolar metabolites with only one injection of the sample for each ionization mode using heart-cutting trapping to combine HILIC and RP separations. This approach enables the trapping of some compounds eluted from the first chromatographic dimension for separation later in the second dimension. In our case, we applied heart-cutting to non-retained metabolites in the first dimension. For that purpose, two independent miniaturized one-dimensional HILIC and RP methods were developed by optimizing the chromatographic and ionization conditions using columns with an inner diameter of 1 mm. They were then merged into one two-dimensional micro LC-MS method by optimization of the trapping conditions. Equilibration of the HILIC column during elution on the RP column and vice versa reduced the overall analysis time, and the multidimensionality allows us to avoid signal measurements during the solvent front. To demonstrate the benefits of this approach to metabolomics, it was applied to the analysis of the human plasma standard reference material SRM 1950, enabling the detection of hundreds of metabolites without the significant loss of some of them while requiring an injection volume of only 0.5 μL.

In comprehensive two-dimensional liquid chromatography (LC × LC), solvents of high eluotropic strength are frequently used in the first dimension ( 1 D), which lead to peak broadening in the second dimension ( 2 D). In the majority of the current LC × LC column combinations, analytes are less than optimally refocused upon transfer to the second column, which negatively affects sensitivity. Furthermore, the typical combination of 1 or 2.1 mm columns in the 1 D paired with a 3 mm (or broader) column in the 2 D leads to at least a 9- or 4-fold dilution and a corresponding loss of sensitivity when using concentration-sensitive detectors. This occurs due to the enhanced radial dilution of the analytes in a broader column, while the sensitivity problem is further exacerbated in LC × LC due to the high flow operated 2 D. In this paper, we introduce a solution to neutralize and inverse this dilution problem through a reconcentrating solution using temperature-responsive liquid chromatography (TRLC) in the 1 D, which is a purely aqueous separation mode. Full solute refocusing at the 2 D column head is thereby obtained when TRLC is combined with reversed-phase liquid chromatography (RPLC). This is shown for the combination of a 2.1 mm I.D. TRLC column with decreasing RPLC column diameters (3-2.1-1 mm) operated at the same linear velocities, hence a resulting decrease in dilution, respectively. Ultraviolet (UV) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) detection were used to determine the experimental detection limits. Sensitivity improvements with UV detection were somewhat lower than expected, but represent ∼1.5- and 3-fold sensitivity enhancement when using a 1 mm I.D. column compared to 2.1 or 3 mm I.D. columns in the 2 D, respectively. This is attributed to extra-column dispersion and the poorer performance of 1 mm I.D. columns. A major benefit of the use of 1 mm I.D. columns in the 2 D is that it allows split-free coupling of 2 D effluent with ESI-MS (at 450 μL/min), making the coupling robust and simple. When using ESI-MS even better, albeit more variable, sensitivity enhancements were obtained on the narrower columns. The benefits of the methodology are demonstrated for paraben test solutes and for phenolic compounds in a blueberry extract by TRLC × RPLC-UV-ESI-TOF-MS.

Two-dimensional liquid chromatography (2D-LC) is a powerful technique used to characterize complex samples such as synthetic polymers, biomacromolecules, and hybrid modalities (conjugates, oligonucleotides, nanoparticles, etc., which fall between traditional small molecules and large molecules). Characterizing such molecules often requires a highly orthogonal 2D-LC workflow to resolve structurally similar impurities. However, it remains a challenge to achieve truly orthogonal 2D-LC coupling due to incompatibility of the chromatographic conditions used in each dimension. In this work, we present a facile strategy of connecting an in-line mixer, in-line mixing modulation (ILMM), to realize challenging 2D-LC workflows: (1) coupling gel permeation chromatography (GPC) with reversed-phase liquid chromatography (RPLC) for hydrophobic oligomer analysis and (2) coupling ion-pair reversed-phase (IPRP) with hydrophilic interaction liquid chromatography (HILIC) for polar antisense oligonucleotide (ASO) analysis. Compared with the state-of-the-art commercially available active solvent modulation (ASM), engaging the ILMM significantly reduces the peak distortion in GPC-RPLC, allowing an at least 67% higher transfer volume from the primary to secondary dimension, and resolves the ASO sample breakthrough in selective comprehensive IPRP×HILIC. Also remarkably, ILMM demonstrated superiority in comprehensive RPLC×RPLC analysis in comparison with ASM, suggesting its potential in broader 2D-LC applications. In addition to chromatography improvement, ILMM offers several advantages over benchmark modulation approaches in regard to alleviating the need of an additional dilution flow and a simple as well as flexible system configuration, opening many opportunities to establish innovative and versatile multidimensional workflows for characterizing compounds with increasing complexity.

The design aspects of microfluidic chips for spatial three-dimensional chromatography featuring an interconnected channel network and targeting protein analysis are discussed, and the corresponding kinetic performance limits have been established using a Pareto-optimality approach. The pros and cons to integrate different separation mechanisms (IEF, CE, SEC, RPLC, HILIC, HIC, and IEX) are discussed considering development stages in the spatial domain ( x LC) in the first and second dimension and time domain ( t LC) for the third dimension. Based on Pareto-optimization, we discuss the considerations of the channel length, particle diameter, and the effect of number of second- and third-dimension channels on the resulting peak capacity of a spatial x IEF × x SEC × t RPLC device. Novel equations are proposed to determine the peak capacity in x SEC and to account for sample modulation affected by the number of second- and third-dimension channels. The corresponding Pareto fronts have been constructed demonstrating the resolving power, in terms of peak capacity and analysis time, considering current state-of-the-art prototyping methodologies. A microfluidic spatial prototype chip with an integrated channel layout (64 2 D and 4096 3 D channels) has been created, which has the potential to yield a peak capacity of 32,600 within only 44 min of the total analysis time, by implementing x IEF × x SEC × t RPLC separation stages.

Sialic acids have diverse biological roles, ranging from promoting up to preventing protein and cellular recognition in health and disease. The various functions of these monosaccharides are owed, in part, to linkage variants, and as a result, linkage-specific analysis of sialic acids is an important aspect of glycomic studies. This has been addressed by derivatization strategies using matrix-assisted laser desorption/ionization mass spectrometry (MS) or sialidase digestion arrays followed by liquid chromatography (LC)-MS. Despite this, these approaches are unable to simultaneously provide unambiguous assignment of sialic acid linkages and assess further isomeric glycan features within a single measurement. Thus, for the first time, we present the combination of procainamide fluorescent labeling with sialic acid linkage-specific derivatization via ethyl esterification and amidation for the analysis of released plasma N -glycans using reversed-phase (RP)LC-fluorescence detection (FD)-MS. As a result, α2,3- and α2,6-sialylated N -glycans, with the same mass prior to derivatization, are differentiated based on retention time, precursor mass, and fragmentation spectra, and additional sialylated isomers were also separated. Furthermore, improved glycan coverage and protocol precision were found via the novel application using a combined FD-MS quantification approach. Overall, this platform achieved unambiguous assignment of N -glycan sialic acid linkages within a single RPLC-FD-MS measurement, and by improving their retention on RPLC, this technique can be used for future investigations of released N -glycans as an additional or orthogonal method to current analytical approaches.

Tandem column liquid chromatography (LC) is a convenient, cost-effective approach to resolve multicomponent mixtures by serially coupling columns on readily available one-dimensional separation systems without specialized user training. Yet, adoption of this technique remains limited, mainly due to the difficulty in identifying optimal selectivity out of many possible tandem column combinations. At this point, method development and optimization require laborious "hit-or-miss" experimentation and "blind" screening when investigating different column selectivity without standard analytes. As a result, many chromatography practitioners end up combining two columns of similar selectivity, limiting the scope and potential of tandem column LC as a mainstay for industrial applications. To circumvent this challenge, we herein introduce a straightforward in silico multifactorial approach as a framework to expediently map the separation landscape across multiple tandem columns (achiral and chiral) and eluent combinations (isocratic and gradient elution) under reversed-phase LC conditions. Retention models were built using commercially available LC simulator software showcasing less than 2% difference between experimental and simulated retention times for analytes of interest in multicomponent pharmaceutical mixtures ( e.g. , metabolites and cyclic peptides).

Adeno-associated viruses (AAVs) are non-enveloped, single-stranded DNA viruses that have recently emerged as an attractive vector for delivering genetic materials to hosts for gene therapy applications. Due to their ability to transduce a wide range of species and tissues in vivo , low risk of immunotoxicity, and mild innate and adaptive immune responses, AAVs are currently used in research and clinical studies as a monotherapy or with other biomolecules to perform gene editing, replacement, addition, and silencing. As AAVs are a new and complex therapeutic modality with molecular weights into the megadalton range, new analytical techniques are therefore needed to support process development, product characterization, and release. In this study, an online two-dimensional liquid chromatography-mass spectrometry (2DLC-MS) method was developed for AAV characterization. Our method uses high-resolution anion-exchange chromatography (AEX) in the first dimension to separate and measure empty and full capsids in AAV samples, followed by reversed-phase liquid chromatography coupled with mass spectrometry (RPLC-MS) to separate and characterize viral proteins. In this technique, online denaturation and removal of MS-incompatible salt were performed following AEX. The viral proteins present in the peak of interest after first-dimensional AEX are subjected to intact protein separation on the second-dimensional RPLC column and then characterized by MS. The 2DLC-MS method demonstrated in this study allows for high-throughput and multi-attribute AAV characterization in a single run, with minimal sample handling required for different AAV serotypes.

Enantioselective chromatography has been the preferred technique for the determination of enantiomeric excess across academia and industry. Although sequential multicolumn enantioselective supercritical fluid chromatography screenings are widespread, access to automated ultra-high-performance liquid chromatography (UHPLC) platforms using state-of-the-art small particle size chiral stationary phases (CSPs) is an underdeveloped area. Herein, we introduce a multicolumn UHPLC screening workflow capable of combining 14 columns (packed with sub-2 μm fully porous and sub-3 μm superficially porous particles) with nine mobile phase eluent choices. This automated setup operates under a vast selection of reversed-phase liquid chromatography, hydrophilic interaction liquid chromatography, polar-organic mode, and polar-ionic mode conditions with minimal manual intervention and high success rate. Examples of highly efficient enantioseparations are illustrated from the integration of chiral screening conditions and computer-assisted modeling. Furthermore, we describe the nuances of in silico method development for chiral separations via second-degree polynomial regression fit using LC simulator (ACD/Labs) software. The retention models were found to be very accurate for chiral resolution of single and multicomponent mixtures of enantiomeric species across different types of CSPs, with differences between experimental and simulated retention times of less than 0.5%. Finally, we illustrate how this approach lays the foundation for a streamlined development of ultrafast enantioseparations applied to high-throughput enantiopurity analysis and its use in the second dimension of two-dimensional liquid chromatography experiments.

Replacement of a non-bridging oxygen atom of the phosphate diester linkage of an oligonucleotide by sulfur conveys pharmacokinetic benefits, such as increased nuclease resistance and enhanced protein binding. Substitution renders the internucleotide linkages chiral, and so phosphorothioate diester (PS) oligonucleotides comprise complex mixtures of diastereoisomers. Currently, chromatographic separation of individual diastereoisomers is limited to oligonucleotides that contain no more than about four or five PS linkages. The development of therapeutic PS oligonucleotides, which often contain >15 PS linkages, would be greatly aided by methods useful for assessing batch-to-batch stereo-reproducibility. To this effect, the relative sensitivities of metal ion complexation chromatography (MICC), in-series reversed phase-strong anion exchange chromatography (RP-SAX), and 31 P NMR toward changes in the diastereoisomeric distributions of therapeutic PS oligonucleotides were compared. Model oligonucleotides synthesized under conditions known to impact PS stereochemistry were used to evaluate the method performance, and all three methods showed excellent sensitivity toward changes in the diastereoisomeric composition. Interactions via the solvent-accessible areas and a combination of hydrophobic and electrostatic forces may be responsible for the selectivity demonstrated by MICC and in-series RP-SAX, respectively.

Machine learning is a popular technique to predict the retention times of molecules based on descriptors. Descriptors and associated labels (e.g., retention times) of a set of molecules can be used to train a machine learning algorithm. However, descriptors are fixed molecular features which are not necessarily optimized for the given machine learning problem (e.g., to predict retention times). Recent advances in molecular machine learning make use of so-called graph convolutional networks (GCNs) to learn molecular representations from atoms and their bonds to adjacent atoms to optimize the molecular representation for the given problem. In this study, two GCNs were implemented to predict the retention times of molecules for three different chromatographic data sets and compared to seven benchmarks (including two state-of-the art machine learning models). Additionally, saliency maps were computed from trained GCNs to better interpret the importance of certain molecular sub-structures in the data sets. Based on the overall observations of this study, the GCNs performed better than all benchmarks, either significantly outperforming them (5-25% lower mean absolute error) or performing similar to them (<5% difference). Saliency maps revealed a significant difference in molecular sub-structures that are important for predictions of different chromatographic data sets (reversed-phase liquid chromatography vs hydrophilic interaction liquid chromatography).

In comparison to proteomics, the application of two-dimensional liquid chromatography (2D LC) in the field of metabolomics is still premature. One reason might be the elevated chemical complexity and the associated challenge of selecting proper separation conditions in each dimension. As orthogonality of dimensions is a major issue, the present study aimed for the identification of successful stationary phase combinations. To determine the degree of orthogonality, first, six different metrics, namely, Pearson's correlation coefficient (1 - | R |), the nearest-neighbor distances ( H̅ NND ), the "asterisk equations" ( A O ), and surface coverage by bins (SC G ), convex hulls (SC CH ), and α-convex hulls (SC αH ), were critically assessed by 15 artificial 2D data sets, and a systematic parameter optimization of α-convex hulls was conducted. SG G generated valid results with sensitivity toward space utilization and data distribution and, therefore, were applied to pairs of experimental retention time sets obtained for >350 metabolites, selected to represent the chemical space of human urine. Normalized retention data were obtained for 23 chromatographic setups, comprising reversed-phase (RP), hydrophilic interaction liquid chromatography (HILIC), and mixed-mode separation systems with an ion exchange (IEX) contribution. As expected, no single LC setting provided separation of all considered analytes, but while conventional RP×HILIC combinations appeared rather complementary than orthogonal, the incorporation of IEX properties into the RP dimension substantially increased the 2D potential. Eventually, one of the most promising column combinations was implemented for an offline 2D LC time-of-flight mass spectrometry analysis of a lyophilized urine sample. Targeted screening resulted in a total of 164 detected metabolites and confirmed the outstanding coverage of the 2D retention space.αH with α = 0.1, and H̅ NND generated valid results with sensitivity toward space utilization and data distribution and, therefore, were applied to pairs of experimental retention time sets obtained for >350 metabolites, selected to represent the chemical space of human urine. Normalized retention data were obtained for 23 chromatographic setups, comprising reversed-phase (RP), hydrophilic interaction liquid chromatography (HILIC), and mixed-mode separation systems with an ion exchange (IEX) contribution. As expected, no single LC setting provided separation of all considered analytes, but while conventional RP×HILIC combinations appeared rather complementary than orthogonal, the incorporation of IEX properties into the RP dimension substantially increased the 2D potential. Eventually, one of the most promising column combinations was implemented for an offline 2D LC time-of-flight mass spectrometry analysis of a lyophilized urine sample. Targeted screening resulted in a total of 164 detected metabolites and confirmed the outstanding coverage of the 2D retention space.

Ultraperformance liquid chromatography (UPLC) and ion mobility (IM) spectrometry were interfaced with mass spectrometry (MS) and tandem mass spectrometry (MS/MS) to characterize a complex nonionic surfactant mixture. The surfactant was composed of a glycerol core, functionalized with poly(ethylene oxide) units (PEO n ) that were partially esterified by caprylic and/or capric acid. Reversed-phase UPLC classified the blend based on polarity into four groups of eluates, corresponding to compounds with zero, one, two, or three fatty acid residues. Additional separation within each eluate group was achieved according to the length of the fatty acid chains. Coeluting molecules of similar polarity were dispersed in the gas phase by their collision cross section in the IM dimension. Performed in series, UPLC and IM allowed for the separation and detection of several isomeric and isobaric blend constituents, thereby enabling their isolation for conclusive MS/MS analysis to confirm or elucidate their primary structures and architectures (overall four-dimensional, 4D, characterization).

Supramolecular hydrogels formed through polymer-nanoparticle interactions are promising biocompatible materials for translational medicines. This class of hydrogels exhibits shear-thinning behavior and rapid recovery of mechanical properties, providing desirable attributes for formulating sprayable and injectable therapeutics. Characterization of hydrogel composition and loading of encapsulated drugs is critical to achieving the desired rheological behavior as well as tunable in vitro and in vivo payload release kinetics. However, quantitation of hydrogel composition is challenging due to material complexity, heterogeneity, high molecular weight, and the lack of chromophores. Here, we present a label-free approach to simultaneously determine hydrogel polymeric components and encapsulated payloads by coupling a reversed phase liquid chromatographic method with a charged aerosol detector (RPLC-CAD). The hydrogel studied consists of modified hydroxypropylmethylcellulose, self-assembled PEG- b -PLA nanoparticles, and a therapeutic compound, bimatoprost. The three components were resolved and quantitated using the RPLC-CAD method with a C4 stationary phase. The method demonstrated robust performance, applicability to alternative cargos ( i.e. , proteins) and was suitable for composition analysis as well as for evaluating in vitro release of cargos from the hydrogel. Moreover, this method can be used to monitor polymer degradation and material stability, which can be further elucidated by coupling the RPLC method with (1) a multi-angle light scattering detector (RPLC-MALS) or (2) high resolution mass spectrometry (RPLC-MS) and a Fourier-transform based deconvolution algorithm. We envision that this analytical strategy could be generalized to characterize critical quality attributes of other classes of supramolecular hydrogels, establish structure-property relationships, and provide rational design guidance in hydrogel drug product development.

To address the complexity of the proteome in mass spectrometry (MS)-based top-down proteomics, multidimensional liquid chromatography (MDLC) strategies that can effectively separate proteins with high resolution and automation are highly desirable. Although various MDLC methods that can effectively separate peptides from protein digests exist, very few MDLC strategies, primarily consisting of 2DLC, are available for intact protein separation, which is insufficient to address the complexity of the proteome. We recently demonstrated that hydrophobic interaction chromatography (HIC) utilizing a MS-compatible salt can provide high resolution separation of intact proteins for top-down proteomics. Herein, we have developed a novel 3DLC strategy by coupling HIC with ion exchange chromatography (IEC) and reverse phase chromatography (RPC) for intact protein separation. We demonstrated that a 3D (IEC-HIC-RPC) approach greatly outperformed the conventional 2D IEC-RPC approach. For the same IEC fraction (out of 35 fractions) from a crude HEK 293 cell lysate, a total of 640 proteins were identified in the 3D approach (corresponding to 201 nonredundant proteins) as compared to 47 in the 2D approach, whereas simply prolonging the gradients in RPC in the 2D approach only led to minimal improvement in protein separation and identifications. Therefore, this novel 3DLC method has great potential for effective separation of intact proteins to achieve deep proteome coverage in top-down proteomics.

Two-dimensional reversed-phase capillary liquid chromatography (2D RPLC) separations have enabled comprehensive proteome profiling of biological systems. However, milligram sample quantities of proteins are typically required due to significant losses during offline fractionation. Such a large sample requirement generally precludes the application samples in the nanogram to low-microgram range. To achieve in-depth proteomic analysis of such small-sized samples, we have developed the nanoFAC (nanoflow Fractionation and Automated Concatenation) 2D RPLC platform, in which the first dimension high-pH fractionation was performed on a 75-μm i.d. capillary column at a 300 nL/min flow rate with automated fraction concatenation, instead of on a typically used 2.1 mm column at a 200 μL/min flow rate with manual concatenation. Each fraction was then fully transferred to the second-dimension low-pH nanoLC separation using an autosampler equipped with a custom-machined syringe. We have found that using a polypropylene 96-well plate as collection device as well as the addition of n -Dodecyl β-d-maltoside (0.01%) in the collection buffer can significantly improve sample recovery. We have demonstrated the nanoFAC 2D RPLC platform can achieve confident identifications of ∼49,000-94,000 unique peptides, corresponding to ∼6,700-8,300 protein groups using only 100-1000 ng of HeLa tryptic digest (equivalent to ∼500-5,000 cells). Furthermore, by integrating with phosphopeptide enrichment, the nanoFAC 2D RPLC platform can identify ∼20,000 phosphopeptides from 100 μg of MCF-7 cell lysate.

Hybrid-supported phospholipid bilayers are a model structure utilized for measurement of molecular interactions that typically occur at cell membranes. These membrane models are prepared by adsorption of a lipid monolayer onto a stable n-alkyl chain layer that is covalently bound to a support surface. Hybrid bilayers have been adapted to chromatographic retention measurements of lipophilicity through the assembly of a phospholipid monolayer onto n-alkane-modified silica surfaces in reversed-phase chromatographic particles. Recent Raman microscopy studies of these particles have shown that the acyl chains of the phospholipid interact with the C 18 -alkyl chains immobilized on the silica surface, where both lipid and C 18 alkyl chains become ordered because of chain interdigitation. Confocal Raman microscopy has also been used to investigate the association of small molecules with hybrid-lipid bilayers in C 18 chromatographic silica particles; the partitioning of model solutes compares favorably to that in lipid vesicle membranes with similar changes in acyl-chain structure (disordering) with solute partitioning. The present study seeks information about how these membrane-mimetic bilayers assemble onto the C 18 -derivatized silica surfaces of reversed-phase chromatographic silica particles. Confocal Raman microscopy is capable of interrogating the time-dependent internal composition and structure within individual silica particles. The Raman scattering data can be resolved into component Raman spectra and corresponding composition vectors that describe the time-dependent changes in intensity of the component spectra. This analysis provides insight into how the structures of both the lipid and C 18 alkyl chains of hybrid lipid bilayers evolve during deposition and organization on the internal surfaces of reversed-phase chromatographic silica particles.

Top-down mass spectrometry (MS)-based proteomics has become a powerful tool for comprehensive characterization of intact proteins. However, because of the high complexity of the proteome, highly effective separation of intact proteins from complex mixtures prior to MS analysis remains challenging. Monolithic columns have shown great promise for intact protein separation due to their high permeability, low backpressure, and fast mass transfer. Herein, for the first time, we developed bridged hybrid bis(triethoxysilyl)ethylene (BTSEY) monolith with C8 functional groups (C8@BTSEY) for highly effective protein separation and coupled it to high-resolution MS for identification of intact proteins from complex protein mixtures. We have optimized mobile phase conditions of our monolith-based reverse-phase chromatography (RPC) for online liquid chromatography (LC)-MS analysis and evaluated separation reproducibility of the C8@BTSEY column. We further assessed the chromatographic performance of this column by separating a complex protein mixture extracted from swine heart tissue. Using our monolithic column (i.d. 100 μm × 35 cm), we separated over 300 proteoforms (up to 104 kDa) from 360 ng of protein mixture in an 80 min one-dimensional (1D) LC run. The highly effective separation and recovery of intact proteins from this monolithic column allowed unambiguous identification of ∼100 proteoforms including a large protein, αactinin2 (103.77 kDa), by online 1D LC-MS/MS analysis for the first time. As demonstrated, this C8@BTSEY column is reproducible and effective in separation of intact proteins, which shows high promise for top-down proteomics.

We have systematically evaluated the chromatographic behavior of post-translationally/chemically modified peptides using data spanning over 70 of the most relevant modifications. These retention properties were measured for standard bottom-up proteomic settings (fully porous C18 separation media, 0.1% formic acid as ion-pairing modifier) using collections of modified/nonmodified peptide pairs. These pairs were generated by spontaneous degradation, chemical or enzymatic treatment, analysis of synthetic peptides, or the cotranslational incorporation of noncanonical proline analogues. In addition, these measurements were validated using external data acquired for synthetic peptides and enzymatically induced citrullination. Working in units of hydrophobicity index (HI, % ACN) and evaluating the average retention shifts (Δ H I) represent the simplest approach to describe the effect of modifications from a didactic point of view. Plotting HI values for modified ( y -axis) vs nonmodified ( x -axis) counterparts generates unique slope and intercept values for each modification defined by the chemistry of the modifying moiety: its hydrophobicity, size, p K a of ionizable groups, and position of the altered residue. These composition-dependent correlations can be used for coarse incorporation of PTMs into models for prediction of peptide retention. More accurate predictions would require the development of specific sequence-dependent algorithms to predict Δ H I values.

Ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the reference separation technique for characterizing oligonucleotides (ONs) and their related impurities. The aim of this study was to better understand the retention mechanism of ONs, evaluate the applicability of the linear solvent strength (LSS) retention model, and explore the potential of ultra-short columns having a length of only 5 mm for the separation of model ONs. First, the validity of the LSS model was evaluated for ONs having sizes comprised between 3 and 30 kDa, and the accuracy of retention time predictions was assessed. It was found that ONs in IP-RPLC conditions follow an "on-off" elution behavior, despite a molecular weight lower than that of proteins. For most linear gradient separation conditions, a column length between 5 and 35 mm was found to be appropriate. Ultra-short columns of only 5 mm were therefore explored to speed up separations by considering the impact of the instrumentation on the efficiency. Interestingly, the impacts of injection volume and post-column connection tubing on peak capacity were found to be negligible. Finally, it was demonstrated that longer columns would not improve selectivity or separation efficiency, but baseline separation of three model ONs mixtures was enabled in as little as 30 s on the 5 mm column. This proof-of-concept work paves the way for future investigations using more complex therapeutic ONs and their related impurities.

The separation mechanism of PEGylated peptides in reversed-phase high-performance liquid chromatography (RP-HPLC) is complex, because the PEGylated molecules exhibit physicochemical properties that are different from those of the parent molecules and have heterogeneous structure. Since most separation studies have focused on the hydrophobicity of the peptide relative to poly(ethylene glycol) (PEG), the role of PEG in the separation of PEGylated peptides on RP-HPLC is not clear. To elucidate the effect of the attached PEG on the retention of PEGylated peptides on RP-HPLC, the mono-PEGylated forms of collagen pentapeptide and octreotide were fractionated drop-by-drop from the outlet of the HPLC system and each drop was subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). This approach demonstrated that the dispersity of the attached PEG leads to the peak broadness of PEGylated peptides in RP-HPLC and the elution order inside the HPLC peak of PEGylated peptides was dependent on the attached PEG chain length. The retention time of PEGylated peptide increased as the attached PEG chain increased in length. When uniform PEG was conjugated to octreotide, its well-resolved positional isomers of the mono-PEGylated forms showed narrow peaks comparable to native peptide peak under the same HPLC conditions, which confirmed the effect of the attached PEG dispersity on the chromatographic behavior of PEGylated peptides. In conclusion, this study indicates that the chromatographic behavior of PEGylated peptides is affected by the PEG chain length dispersity as well as the peptide's hydrophobicity.

Oxidized glycerophosphoethanolamines (oxPEs) represent a subclass of bioactive lipids that have intricate roles in various physiological and pathological events. Conventional mass spectrometric methods cannot provide unambiguous information to locate the OH group and the sites of unsaturation. Herein, we report a combined strategy for in-depth structural characterization of oxPEs, including radical-directed dissociation tandem mass spectrometry (RDD-MS/MS) for localizing the OH group and the Paternò-Büchi derivatization coupled with tandem mass spectrometry for pinpointing carbon-carbon double-bond locations. The RDD-MS/MS method has been integrated on a reversed-phase liquid chromatography-mass spectrometry workflow. It enables the profiling of 24 distinct oxPE molecules with unequivocal assignment of the OH sites at nM sensitivity in bovine liver lipid extract treated by soybean 15-lipoxygenase. These findings showcase that the developed method has a good potential in analyzing biological systems where oxPEs may play important roles.

Changes in anthocyanin chemistry represent some of the most important transformations involved in red wine aging. However, accurate analysis of the derived pigments, as required to study the evolution of anthocyanins and tannins during aging, is hampered by their extreme structural diversity, low levels, and the fact that many of these compounds have identical mass spectral characteristics. In this context, chromatographic separation is critical. In this contribution, the application of online hydrophilic interaction chromatography (HILIC) × reversed-phase liquid chromatography (RP-LC) separation coupled to high-resolution mass spectrometry (MS) is described for the detailed characterization of anthocyanins and their derived pigments in aged red wine. A systematic approach was followed for the optimization of HILIC × RP-LC separation parameters using a capillary liquid chromatography (LC) system in the first dimension and an ultrahigh-pressure LC system in the second dimension to ensure maximum sensitivity and performance. Ninety four (94) anthocyanin-derived pigments were tentatively identified in one- and six-year-old Pinotage wines using accurate mass and fragmentation information obtained using quadrupole-time-of-flight mass spectrometry (Q-TOF-MS). Online HILIC × RP-LC-MS was found to offer high-resolution separation, because of the combination of two different separation modes, while the structured elution order observed improved the certainty in compound identification. Therefore, this approach shows promise for the detailed elucidation of the chemical alteration of anthocyanins during wine aging.

Protein tyrosine nitration (PTN) is a signature hallmark of radical-induced nitrative stress in a wide range of pathophysiological conditions, with naturally occurring abundances at substoichiometric levels. In this present study, a fully automated four-dimensional platform, consisting of high-/low-pH reversed-phase dimensions with two additional complementary, strong anion (SAX) and cation exchange (SCX), chromatographic separation stages inserted in tandem, was implemented for the simultaneous mapping of endogenous nitrated tyrosine-containing peptides within the global proteomic context of a Macaca fascicularis cerebral ischemic stroke model. This integrated RP-SA(C)X-RP platform was initially benchmarked through proteomic analyses of Saccharomyces cerevisiae, revealing extended proteome and protein coverage. A total of 27 144 unique peptides from 3684 nonredundant proteins [1% global false discovery rate (FDR)] were identified from M. fascicularis cerebral cortex tissue. The inclusion of the S(A/C)X columns contributed to the increased detection of acidic, hydrophilic, and hydrophobic peptide populations; these separation features enabled the concomitant identification of 127 endogenous nitrated peptides and 137 transmembrane domain-containing peptides corresponding to integral membrane proteins, without the need for specific targeted enrichment strategies. The enhanced diversity of the peptide inventory obtained from the RP-SA(C)X-RP platform also improved analytical confidence in isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analyses.

The market of protein therapeutics is exploding, and characterization methods for proteins are being further developed to understand and explore conformational structures with regards to function and activity. There are several spectroscopic techniques that allow for analyzing protein secondary structure in solution. However, a majority of these techniques need to use purified protein, concentrated enough in the solution to produce a relevant spectrum. In this study, we describe a novel approach which uses ultrahigh pressure liquid chromatography (UHPLC) coupled with mass-spectrometry (MS) to explore compressibility of the secondary structure of proteins under increasing pressure detected by hydrogen-deuterium exchange (HDX). Several model proteins were used for these studies. The studies were conducted with UHPLC in isocratic mode at constant flow rate and temperature. The pressure was modified by a backpressure regulator up to about 1200 bar. It was found that the increase of retention factors upon pressure increase, at constant flow rate and temperature, was based on reduction of the proteins' molecular molar volume. The change in the proteins' molecular molar volume was caused by changes in protein folding, as was revealed by differential deuterium exchange. The degree of protein folding under certain UHPLC conditions can be controlled by pressure, at constant temperature and flow rate. By modifying pressure during UHPLC separation, it was possible to achieve changes in protein folding, which were manifested as changes in the number of labile protons exchanged to deuterons, or vice versa. Moreover, it was demonstrated with bovine insulin that a small difference in the number of protons exchanged to deuterons (based on protein folding under pressure) could be observed between batches obtained from different sources. The use of HDX during UHPLC separation allowed one to examine protein folding by pressure at constant flow rate and temperature in a mixture of sample solution with minimal amounts of sample used for analysis.

The mounting complexity of new modalities in the biopharmaceutical industry entails a commensurate level of analytical innovations to enable the rapid discovery and development of novel therapeutics and vaccines. Hydrophobic interaction chromatography (HIC) has become one of the widely preferred separation techniques for the analysis and purification of biopharmaceuticals under nondenaturing conditions. Inarguably, HIC method development remains very challenging and labor-intensive owing to the numerous factors that are typically optimized by a "hit-or-miss" strategy (e.g., the nature of the salt, stationary phase chemistry, temperature, mobile phase additive, and ionic strength). Herein, we introduce a new HIC method development framework composed of a fully automated multicolumn and multieluent platform coupled with in silico multifactorial simulation and integrated fraction collection for streamlined method screening, optimization, and analytical-scale purification of biopharmaceutical targets. The power and versatility of this workflow are showcased by a wide range of applications including trivial proteins, monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), oxidation variants, and denatured proteins. We also illustrate convenient and rapid HIC method development outcomes from the effective combination of this screening setup with computer-assisted simulations. HIC retention models were built using readily available LC simulator software outlining less than a 5% difference between experimental and simulated retention times with a correlation coefficient of >0.99 for pharmaceutically relevant multicomponent mixtures. In addition, we demonstrate how this approach paves the path for a straightforward identification of first-dimension HIC conditions that are combined with mass spectrometry (MS)-friendly reversed-phase liquid chromatography (RPLC) detection in the second dimension (heart-cutting two-dimensional (2D)-HIC-RPLC-diode array detector (DAD)-MS), enabling the analysis and purification of biopharmaceutical targets.

Octadecanoids are broadly defined as oxylipins ( i.e., lipid mediators) derived from 18-carbon fatty acids. In contrast to the well-studied eicosanoids, there is a lack of analytical methods for octadecanoids, hampering further investigations in the field. We developed an integrated workflow combining chiral separation by supercritical fluid chromatography (SFC) and reversed-phase liquid chromatography (LC) coupled to tandem mass spectrometry detection for quantification of a broad panel of octadecanoids. The platform includes 70 custom-synthesized analytical and internal standards to extend the coverage of the octadecanoid synthetic pathways. A total of 103 octadecanoids could be separated by chiral SFC and complex enantioseparations could be performed in <13 min, while the achiral LC method separated 67 octadecanoids in 13.5 min. The LC method provided a robust complementary approach with greater sensitivity relative to the SFC method. Both methods were validated in solvent and surrogate matrix in terms of linearity, lower limits of quantification (LLOQ), recovery, accuracy, precision, and matrix effects. Instrumental linearity was good for both methods ( R 2 > 0.995) and LLOQ ranged from 0.03 to 6.00 ng/mL for SFC and 0.01 to 1.25 ng/mL for LC. The average accuracy in the solvent and surrogate matrix ranged from 89 to 109% in SFC and from 106 to 220% in LC, whereas coefficients of variation (CV) were <14% (at medium and high concentrations) and 26% (at low concentrations). Validation in the surrogate matrix showed negligible matrix effects (<16% for all analytes), and average recoveries ranged from 71 to 83%. The combined methods provide a platform to investigate the biological activity of octadecanoids and expand our understanding of these little-studied compounds.

The elution profile of chromatographic peaks provides fundamental understanding of the processes that occur in the mobile phase and the stationary phase. Major advances have been made in the column chemistry and suppressor technology in ion chromatography (IC) to handle a variety of sample matrices and ions. However, if the samples contain high concentrations of matrix ions, the overloaded peak elution profile is distorted. Consequently, the trace peaks shift their positions in the chromatogram in a manner that depends on the peak shape of the overloading analyte. In this work, the peak shapes in IC are examined from a fundamental perspective. Three commercial IC columns AS16, AS18, and AS23 were studied with borate, hydroxide and carbonate as suppressible eluents. Monovalent ions (chloride, bromide, and nitrate) are used as model analytes under analytical (0.1 mM) to overload conditions (10-500 mM). Both peak fronting and tailing are observed. On the basis of competitive Langmuir isotherms, if the eluent anion is more strongly retained than the analyte ion on an ion exchanger, the analyte peak is fronting. If the eluent is more weakly retained on the stationary phase, the analyte peak always tails under overload conditions regardless of the stationary phase capacity. If the charge of the analyte and eluent anions are different (e.g., Br(-) vs CO3(2-)), the analyte peak shapes depend on the eluent concentration in a more complex pattern. It was shown that there are interesting similarities with peak distortions due to strongly retained mobile phase components in other modes of liquid chromatography.

Due to its high specific surface area and chemical stability, porous silica is used as a support structure in numerous applications, including heterogeneous catalysis, biomolecule immobilization, sensors, and liquid chromatography. Reversed-phase liquid chromatography (RPLC), which uses porous silica support particles, has become an indispensable separations tool in quality control, pharmaceutics, and environmental analysis requiring identification of compounds in mixtures. For complex samples, the need for higher resolution separations requires an understanding of the time scale of processes responsible for analyte retention in the stationary phase. In the present work, single-molecule fluorescence imaging is used to observe transport of individual molecules within RPLC porous silica particles. This technique allows direct measurement of intraparticle molecular residence times, intraparticle diffusion rates, and the spatial distribution of molecules within the particle. On the basis of the localization uncertainty and characteristic measured diffusion rates, statistical criteria were developed to resolve the frame-to-frame behavior of molecules into moving and stuck events. The measured diffusion coefficient of moving molecules was used in a Monte Carlo simulation of a random-walk model within the cylindrical geometry of the particle diameter and microscope depth-of-field. The simulated molecular transport is in good agreement with the experimental data, indicating transport of moving molecules in the porous particle is described by a random-walk. Histograms of stuck-molecule event times, locations, and their contributions to intraparticle residence times were also characterized.

Isolation and chemical characterization of target components in fast-paced pharmaceutical laboratories can often be challenging, especially when dealing with mixtures of closely related, possibly unstable species. Traditionally, this process involves intense labor and manual intervention including chromatographic method development and optimization, fraction collection, and drying processes prior to NMR analyses for unambiguous structure elucidation. To circumvent these challenges, a foundational framework for the proper utilization of supercritical carbon dioxide (scCO 2 ) and deuterated modifiers (CD 3 OD) in sub/supercritical fluid chromatography (SFC) is herein introduced. This facilitates a streamlined multicomponent isolation with minimized protic residues, further enabling immediate NMR analysis. In addition to bypassing tedious drying processes and minimizing analyte degradation, this approach (complementary to traditional reversed-phase liquid chromatography, RPLC) delivers highly efficient separations and automated fraction collection using readily available analytical/midscale SFC instrumentation. A series of diverse analytes across a wide spectrum of chemical properties (acid, basic, and neutral), combined with different stationary-phase columns in SFC are investigated using both a protic organic modifier (CH 3 OH) and its deuterated counterpart (CD 3 OD). The power of this framework is demonstrated with pharmaceutically relevant applications in the context of target characterization and analysis of complex multicomponent reaction mixtures from modern synthetic chemistry, demonstrating high isolation yields while reducing both the environmental footprint and manual intervention. This workflow enables unambiguous fast-paced structure elucidation on the analytical scale, providing results that are comparable to traditional, but time-consuming, RPLC purification approaches.

An integrated sample treatment device, composed of a membrane interface and a monolithic hybrid silica based immobilized enzymatic reactor (IMER), was developed for the simultaneous sample buffer exchange, protein enrichment, and online digestion, by which for the sample buffer, the acetonitrile content was reduced to approximately 1/10 of the initial one, and the pH value was adjusted from approximately 3.0 to approximately 8.0, compatible for online trypsin digestion. Furthermore, the signal intensity of myoglobin digests was improved by over 10 times. Such an integrated device was successfully applied to the online treatment of three protein eluates obtained by reverse-phase liquid chromatography (RPLC) separation, followed by further protein digest analysis with microreverse-phase liquid chromatography-electrospray ionization-tandem mass spectrometry (microRPLC-ESI-MS/MS). The experimental results showed that the performance of such an integrated sample treatment device was comparable to that of the traditional offline sample treatment method, including lyophilization and in-solution digestion. However, the consumed time was reduced to 1/192. All these results demonstrate that such an integrated sample treatment device could be further online coupled with protein separation, peptide separation, and identification, to achieve high-throughput proteome analysis.

SARS-CoV-2 cellular infection is mediated by the heavily glycosylated spike protein. Recombinant versions of the spike protein and the receptor-binding domain (RBD) are necessary for seropositivity assays and can potentially serve as vaccines against viral infection. RBD plays key roles in the spike protein's structure and function, and thus, comprehensive characterization of recombinant RBD is critically important for biopharmaceutical applications. Liquid chromatography coupled to mass spectrometry has been widely used to characterize post-translational modifications in proteins, including glycosylation. Most studies of RBDs were performed at the proteolytic peptide (bottom-up proteomics) or released glycan level because of the technical challenges in resolving highly heterogeneous glycans at the intact protein level. Herein, we evaluated several online separation techniques: (1) C2 reverse-phase liquid chromatography (RPLC), (2) capillary zone electrophoresis (CZE), and (3) acrylamide-based monolithic hydrophilic interaction chromatography (HILIC) to separate intact recombinant RBDs with varying combinations of glycosylations (glycoforms) for top-down mass spectrometry (MS). Within the conditions we explored, the HILIC method was superior to RPLC and CZE at separating RBD glycoforms, which differ significantly in neutral glycan groups. In addition, our top-down analysis readily captured unexpected modifications (e.g., cysteinylation and N-terminal sequence variation) and low abundance, heavily glycosylated proteoforms that may be missed by using glycopeptide data alone. The HILIC top-down MS platform holds great potential in resolving heterogeneous glycoproteins for facile comparison of biosimilars in quality control applications.

The increased presence of synthetic trans fatty acids into western diets has been shown to have deleterious effects on physiology and raising an individual's risk of developing metabolic disease, cardiovascular disease, and stroke. The importance of these fatty acids for health and the diversity of their (patho) physiological effects suggest that not only should the free trans fatty acids be studied but also monitoring the presence of these fats into the side chains of biological lipids, such as glycerophospholipids, is also essential. We developed a high resolution LC-MS method that quantitatively monitors the major lipid classes found in biospecimens in an efficient, sensitive, and robust manner while also characterizing individual lipid side chains through the use of high energy collisional dissociation (HCD) fragmentation and chromatographic alignment. We herein show how this previously described reversed phase method can baseline separate the cis-trans isomers of phosphatidylglycerol and phosphatidylcholine (PC) with two 18:1 side chains, in both positive and negative mode, as neat solutions and when spiked into a biological matrix. Endogenous PC (18:1/18:1)-cis and PC (18:1/18:1)-trans isomers were examined in mitochondrial and serum profiling studies, where rats were fed diets enriched in either trans 18:1 fatty acids or cis 18:1 fatty acids. In this study, we determined the cis:trans isomer ratios of PC (18:1/18:1) and related this ratio to dietary composition. This generalized LC-MS method enables the monitoring of trans fats in biological lipids in the context of a nontargeted method, allowing for relative quantitation and enhanced identification of unknown lipids in complex matrixes.

We have developed a new SERS substrate based on the reduction of silver nitrate in the presence of ZnS-capped CdSe quantum dots. This substrate showed higher sensitivities as compared to a hydroxylamine-reduced silver sol. On the basis of this new substrate, at-line SERS detection was coupled with capillary liquid chromatography (cap-LC) for the separation and selective determination of pyrimidine and purine bases. For this purpose, wells of a dedicated microtiter plate were loaded with 20 μL of the SERS substrate and placed on an automated x,y translation stage. A flow-through microdispenser capable of ejecting 50 pL droplets, at a frequency 100 Hz, was used as the interface to connect the cap-LC system to the wells loaded with SERS substrate. A detailed study of the dependence of both the separation and the surface-enhanced Raman spectra of each base on the pH was performed to optimize the system for maximum sensitivity and selectivity. Highly satisfactory analytical figures of merit were obtained for the six investigated bases (cytosine, xanthine, hypoxanthine, guanine, thymine, and adenine) with detection limits ranging between 0.2 and 0.3 ng injected on the capillary LC column, and the precisions were in the range of 3.0-6.3%.

The compatibility of superficially porous (SP) resin for label-free intact protein analysis with online capillary LC/MS is demonstrated to give improved chromatographic resolution, sensitivity, and reproducibility. The robustness of the platform was measured against several samples of varying complexity and sample loading amount. The results indicate that capillary SP columns provide high loading capacities and that ∼6 s chromatographic peak widths are typical for standard proteins in simple mixtures and proteins isolated from cell and tissue lysates. Subfemtomole detection limits for standard proteins were consistently observed, with the lowest levels at 12 amol for ubiquitin. The analysis of total heart homogenates shows that capillary SP columns provide theoretical peak capacity of 106 protein forms with 30 min total analysis time and enabled detection of proteins from complex mixtures with a single high-resolution scan. The SPLC/MS platform also detected 343 protein forms from two HeLa acid extract replicate analyses that consumed 5 × 10(4) cells and 30 min analysis time, each. Comparison of all the species observed in each HeLa replicate showed 90% overlap (309 forms) with a Pearson correlation coefficient of 89.9% for the common forms observed in the replicates. Efficient acid extract of 1 × 10(4) HeLa cells allowed reproducible detection of common modification states and members from all five of the histone families and demonstrated that capillary SPLC/MS supports reproducible label-free profiling of histones in <15 min total analysis time. The data presented demonstrate that a capillary LC/MS platform utilizing superficially porous stationary phase and a LTQ-Orbitrap FT-MS is fast, sensitive, and reproducible for intact protein profiling from small tissue and cell amounts.

In eukaryotes, actual DNA methylation patterns provide biologically important information, for which both, genome-wide and locus-specific methylation at cytosine residues have been extensively studied. The original contribution of this work relies on the selective derivatization of cytosine moieties with 2-bromoacetophenone for the determination of global DNA methylation by reversed phase high performance liquid chromatography with spectrofluorimetric detection. The important features of the proposed procedure are as follows: (1) no need for the elimination of RNA, (2) detection limits for cytidine, 2'-deoxycytidine, 5-methylcytidine, and 5-methyl-2'-deoxycytidine in the range of 14.4-22.7 fmol, (3) feasibility for the detection of 0.06% of methylation in a low amount of DNA (80 ng), (4) potential viability for the evaluation of RNA methylation, and (5) relative simplicity in terms of analytical instrumentation and personnel training. The results obtained in the analysis of salmon testes DNA and nucleic acids from plant, human blood, and earthworms demonstrate the utility of the proposed procedure in biological studies and, in particular, for evaluation of the potential effect of environmental factors on actual DNA methylation in different types of living organisms., (© 2011 American Chemical Society)

Although it has been known for decades that arsenic forms fat-soluble arsenic compounds, only recent attempts to identify the compounds have been successful by using a combination of fractionation and elemental and molecular mass spectrometry. Here we show that arsenolipids can directly be identified and quantified in biological extracts using reversed-phase high-performance liquid chromatography (RP-HPLC) simultaneously online-coupled to high-resolution inductively coupled plasma mass spectrometry (ICPMS) and high-resolution electrospray mass spectrometry (ES-MS) without having a lipophilic arsenic standard available. Using a methanol gradient for the separation made it necessary to use a gradient-dependent arsenic response factor for the quantification of the fat-soluble arsenic species in the extract. The response factor was obtained by using the ICPMS signal of known concentration of arsenic. The arsenic response was used to determine species-specific response factors for the different arsenic species. The retention time for the arsenic species was utilized to mine the ES-MS data for accurate mass and their tandem mass spectrometry (MS/MS) fragmentation pattern to give information of molecular formula and structure information. The majority of arsenolipids, found in the hexane phase of fish meal from capelin ( Mallotus villosus ) was in the form of three dimethylarsinoyl hydrocarbons (C(23)H(38)AsO, C(17)H(38)AsO, C(19)H(42)AsO) with minor amounts of dimethylarsinoyl fatty acids (C(17)H(36)AsO(3), C(23)H(38)AsO(3), C(24)H(38)AsO(3)). One of the dimethylarsinoyl fatty acids (C(24)H(38)AsO(3)), with an even number of carbon in the fatty acid chain, was identified for the first time in this work. This molecular formula is unusual and in contrast to all previously identified arsenic-containing fatty acids with odd numbers of carbon.

In this article, we introduce a method using nanoscale ion-pair reversed-phase high-performance liquid chromatography (nano-IP-RP-HPLC) hyphenated to nanoelectrospray ionization high-resolution mass spectrometry (nano-ESI-HRMS) to separate and identify metabolites in cell extracts. Separation of metabolites was performed on a 100 μm i.d. C18 column with tributylamine (TBA) as the ion-pairing reagent and methanol as the eluent. Basic pH (9.4) of the mobile phase was critical to achieve sufficient retention and sharp metabolite elution at a low concentration of TBA (1.7 mM). Limits of detection were determined for 54 standards with an LTQ-Orbitrap mass spectrometer to be in the upper attomole to low femtomole range for key metabolites such as nucleotides, phosphorylated sugars, organic acids, and coenzyme A thioesters in solvent as well as in a complex matrix. To further evaluate the method, metabolome analysis was performed injecting different amounts of biomass of the methylotroph model organism Methylobacterium extorquens AM1. A (12)C/(13)C labeling strategy was implemented to improve metabolite identification. Analysis of three biological replicates performed with 1.5 ng of cell dry weight biomass equivalents resulted in the identification of 20 ± 4 metabolites, and analysis of 150 ng allowed identifying 157 ± 5 metabolites from a large spectrum of metabolite classes.

A microfluidic device capable of two-dimensional reversed-phase liquid chromatography-capillary electrophoresis with integrated electrospray ionization (LC-CE-ESI) for mass spectrometry (MS)-based proteomic applications is described. Traditional instrumentation was used for the LC sample injection and delivery of the LC mobile phase. The glass microfabricated device incorporated a sample-trapping region and an LC channel packed with reversed-phase particles. Rapid electrokinetic injections of the LC effluent into the CE dimension were performed at a cross-channel intersection. The CE separation channel terminated at a corner of the square device, which functioned as an integrated electrospray tip. In addition to LC-CE-ESI, this device was used for LC-ESI without any instrumental modifications. To evaluate the system, LC-MS and LC-CE-MS analyses of protein digests were performed and compared.

A facile integrated platform for proteome profiling was established, in which native proteins were online denatured and reduced within a heater, digested with an immobilized trypsin microreactor, and analyzed by microflow reversed-phase liquid chromatography with electrospray ionization tandem mass spectrometry (microRPLC-ESI-MS/MS). In comparison to the traditional off-line urea denaturation protocol, even more unique peptides were obtained by online heating in triplicate (14 +/- 2 vs 11 +/- 2 for myoglobin and 16 vs 12 +/- 1 for BSA) within a significantly shortened pretreatment time of approximately 3.5 min (including 1 min of thermal denaturation and reduction and approximately 2.5 min of microreactor digestion). Moreover, proteins with concentrations ranging from 50 ng/mL (approximately 6 fmol) to 1 mg/mL (approximately 120 pmol) were positively identified by the online system. Such a platform was further successfully applied for analyzing the soluble fraction of mouse liver extract. Of all the 367 proteins identified from samples pretreated by the urea protocol and online heating, approximately 40% were overlapped, showing the partial complementation of both approaches. All these results demonstrate that the online integrated platform is of great promise for high-throughput proteome profiling and improved identification capacity for low-abundance proteins with a minute sample amount.

We have developed a one-step method to isolate protein C-terminal peptides from V8 protease-digested proteins by metal oxide-based ligand-exchange (MOLEX) chromatography. V8 protease cleaves the C-terminal side of Asp and Glu, affording a digested peptide with two carboxy groups at the C-terminus, whereas the protein C-terminal peptide has only one α-carboxy group. In MOLEX chromatography, a stable chelate is formed between dicarboxylates and metal atoms, so that the nonterminal (i.e., internal) peptide is retained, whereas the protein C-terminal peptide flows through the MOLEX column. After the optimization of the MOLEX chromatographic conditions, 1619 protein C-termini were identified from 30 μg of peptides (10 μg each, in triplicate) derived from human HeLa cells by means of nanoLC/MS/MS. When the MOLEX-isolated sample from 200 μg of HeLa peptides was further divided into six fractions by high-pH reversed-phase liquid chromatography (LC) prior to nanoLC/MS/MS, 2203 protein C-termini were identified with less than 3% contamination with internal peptides. We believe that this is the largest coverage with the highest purity reported to date in human protein C-terminomics. This fast, simple, sensitive, and selective method to isolate protein C-terminal peptides should be useful for profiling protein C-termini on a proteome-wide scale.

Untargeted metabolomics is an essential component of systems biology research, but it is plagued by a high proportion of detectable features not identified with a chemical structure. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) experiments produce spectra that can be searched against databases to help identify or classify these unknowns, but many features do not generate spectra of sufficient quality to enable successful annotation. Here, we explore alterations to gradient length, mass loading, and rolling precursor ion exclusion parameters for reversed phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) that improve compound identification performance for human plasma samples. A manual review of spectral matches from the HILIC data set was used to determine reasonable thresholds for search score and other metrics to enable semi-automated MS/MS data analysis. Compared to typical LC-MS/MS conditions, methods adapted for compound identification increased the total number of unique metabolites that could be matched to a spectral database from 214 to 2052. Following data alignment, 68.0% of newly identified features from the modified conditions could be detected and quantitated using a routine 20-min LC-MS run. Finally, a localized machine learning model was developed to classify the remaining unknowns and select a subset that shared spectral characteristics with successfully identified features. A total of 576 and 749 unidentified features in the HILIC and RPLC data sets were classified by the model as high-priority unknowns or higher-importance targets for follow-up analysis. Overall, our study presents a simple strategy to more deeply annotate untargeted metabolomics data for a modest additional investment of time and sample.

Top-down proteomics (TDP) is an ideal approach for deciphering the histone code and it routinely employs reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS). Because of the extreme complexity of histones regarding the number of proteoforms, new analytical tools with high-capacity separation and highly sensitive detection of proteoforms are required for TDP of histones. Here we present capillary zone electrophoresis (CZE)-MS/MS via the electro-kinetically pumped sheath-flow CE-MS interface for large-scale top-down delineation of histone proteoforms. CZE-MS/MS identified a comparable number of proteoforms to RPLC-MS/MS from a calf histone sample with more than 30-fold less sample consumption (75-ng vs. Three μg), indicating its substantially higher sensitivity. We identified about 400 histone proteoforms from the calf histone sample using two-dimensional size-exclusion chromatography (SEC)-CZE-MS/MS with less than 300-ng proteins consumed. We identified histone proteoforms carrying various tentative post-translational modifications (PTMs), for example, acetylation, methylation (mono-, di-, and tri-), phosphorylation, and succinylation. The electrophoretic mobility (μ ef ) of unmodified histone proteoforms can be predicted accurately ( R 2 = 0.98) with an optimized semiempirical model based on our recent work. The results render CZE-MS/MS as a useful tool for deciphering the histone code in a proteoform-specific manner and on a global scale.