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Adenovirus (AdVs) is the viral vector of choice in vaccines and oncolytic applications owing to its high transduction activity and inherent immunogenicity. For decades, AdV isolation has relied on ultracentrifugation and ion-exchange chromatography, which are not suitable to large-scale production and struggle to deliver sufficient purity. Immunoaffinity chromatography resins of recent introduction feature high binding capacity and selectivity, but mandate harsh elution conditions (pH 3.0), afford low yield (< 20%), and provide limited reusability. Seeking a more efficient and affordable alternative, this study introduces the first peptide affinity ligands for AdV purification. The peptides were identified via combinatorial selection and in silico design to target hexons, the most abundant proteins in the adenoviral capsid. Selected peptide ligands AEFFIWNA and TNDGPDYSSPLTGSG were conjugated on chromatographic resins and utilized to purify AdV serotype 5 from HEK293 and Vero cell lysates. The peptide-functionalized resins feature high binding capacity (> 10 10 active virions per mL at the residence time of 2 min), provide high yield (> 50%) and up to 100-fold reduction of host cell proteins and DNA. Notably, the peptide ligands enable gentle elution conditions (pH 8) that prevent the "shedding" of penton and fiber proteins, thus affording intact adenovirus particles with high cell-transduction activity. The study of the peptide ligands by surface plasmon resonance and molecular docking and dynamics simulations confirmed the selective targeting of hexon proteins and elucidated the molecular-level mechanisms underlying binding and release. Collectively, these results demonstrate the strong promise of peptide ligands presented herein for the affinity purification of AdVs from cell lysates., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Adeno-associated viruses (AAVs) have emerged as a prominent family of vectors for gene delivery, providing therapeutic options to diseases once deemed incurable. At the same time, they necessitate efficient and affordable purification methods that can be platformed to serve all AAV serotypes. Current chromatographic tools, while affording high product purity, fail to bind certain serotypes, provide limited yield and lifetime, and impose harsh elution conditions that can compromise the vector's activity and safety. Addressing these challenges, this work demonstrates the application of new peptide ligands as the first serotype-agnostic technology for AAV purification by affinity chromatography. Our study reveals a pH-dependent affinity interaction: AAV2, AAV3, AAV6, AAV9, and AAVrh.10 are effectively captured at neutral pH, while binding AAV1, AAV5, AAV7, and AAV8 is stronger in a slightly acidic environment. The elution of bound AAVs was achieved using magnesium chloride at neutral pH for all serotypes, consistently affording capsid yields above 50% and genome yields above 80%, together with a >100-fold reduction in host cell proteins and nucleic acids. In particular, peptide ligand A10 exhibited remarkable binding capacity (> 10 14 vp per mL of resin) and purification performance for all AAV serotypes, demonstrating broad applicability for gene therapy manufacturing. Finally, this work introduces novel alkaline-stable variants of A10 and demonstrates their use as the first affinity ligands capable of performing multiple cycles of AAV2, AAV8, and AAV9 purification with intermediate caustic cleaning without loss of capacity or product quality. Collectively, these results demonstrate the promise of this technology to further the impact and affordability of gene therapy., Competing Interests: Declaration of competing interest Eduardo Barbieri, Michael Crapanzano, and Stefano Menegatti declare conflict of interest respectively as Senior Scientist, Chief Executive Officer, and Chief Technology Officer of LigaTrap Technologies. The authors Stefano Menegatti and Shriarjun Shastry are the authors of Patent Application PCT/US24/29159, titled "Compositions and Methods for Purifying Viral Vectors", filed on May 13 2024 at 07:56:57 pm (Eastern Time). The application was filed by Peter J. Schlueter at Casimir Jones, S.C. (address: 2275 Deming Way, Suite 310 Middleton, WI 53562, US; email: pjschlueter@casimirjones.com) on behalf of North Carolina State University as the applicant. The invention has been exclusively licensed to LigaTrap Technologies, LLC. All other authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In this study, the enzymatic hydrolysates of skipjack tuna, Katsuwonus pelamis, were purified by ultrafiltration and further identified through micro-ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (micro-UPLC-QTOF-MS). The potential umami peptides were identified using both conventional collision-induced dissociation (CID) and novel electron-activated dissociation (EAD) fragmentation techniques. Nine novel umami peptides with iUmami-SCM > 588 were screened. Sensory evaluation and electronic tongue analysis were performed to confirm the taste characteristics of the umami peptides, indicating that these umami peptides all exhibited varying degrees of umami taste. Molecular docking and molecular dynamics simulation were utilized to investigate the interaction with T1R1/T1R3 taste receptors. The docking results revealed that Asp234, Ser23, Glu231, and Ile237 appeared most frequently in all docking sites and formed stable complexes through hydrogen bonding and electrostatic interactions. Furthermore, molecular dynamics simulation allowed for a more comprehensive analysis of their interactions within a dynamic environment, providing a deeper understanding of the umami perception mechanism involving umami peptides and receptors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The retention of three peptides was studied under analytical and overloaded conditions at different concentrations of trifluoroacetic acid (TFA) and water added to the co-solvent methanol (MeOH). Four columns with different stationary phase properties, i.e., silica, diol, 2-ethylpyridine and cyanopropyl (CN) columns, were evaluated in this investigation. The overall aim was to get a deeper understanding on how column chemistry as well as water and TFA in the co-solvent affect the analytical and overloaded elution profiles using multivariate design of experiments and adsorption measurements of co-solvent components. Multivariate experimental design modeling indicated that water had on average around five times higher effect on the retention than the addition of TFA. The results also showed that the retention increases with the addition of TFA and water to the co-solvent on all columns except the CN column, on which the retention decreased. When examining the effect of adding water to the co-solvent, evidence of a hydrophilic interaction liquid chromatography (HILIC)-like retention mechanism was found on the three other columns with more polar stationary phases. However, on the CN column water acted as an additive, decreasing the retention due to competition with the peptide for available adsorption surface. Adsorption isotherm measurements of the polar solvent MeOH showed that MeOH adsorbs much weaker on the CN column than on the other columns. Addition of TFA and water to the co-solvent substantially sharpened the elution profiles under both overloaded and analytical conditions. Adding a small amount of TFA (from 0 % to 0.05 %) to the co-solvent substantially improved the peak shape of the elution profiles, while further addition (from 0.05 % to 0.15 %) had only a minor effect on the elution profile shape. The reduced retention on the CN column could not be explained by TFA adsorption, which was very weak on all studied columns (retention factor, 0.05-0.15). One could therefore speculate that the ion-pairing complex formed between the peptide and TFA in the mobile phase, reduce the retention due to its reduced polarity. On the other columns displaying HILIC-like properties, the TFA probably just decreased the pH of the mobile phase, thereby promoting the partitioning of the peptide into the water-rich layer. Finally, peak deformation due to diluent-eluent mismatch was observed under overloaded conditions. This was most severe in the cases where MeOH adsorption to the stationary phase was strong and the peptides were only mildly retained. Adding 1,4-dioxan to the diluent resolved this issue., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The precise determination of polypeptide antibiotics (PPTs) in foods has been always challenging because of the interference of various endogenous peptides in complex matrix. Herin, a novel large-pore covalent organic framework (TABPT-SPDA-COF) with accessible pore size of 7.9 nm was synthesized as a solid phase extraction (SPE) absorbent for efficiently enriching four PPTs existed in foods originating from animals. The parameters of SPE process were systematically optimized. Subsequently, four PPTs were determined by UHPLC-MS/MS. Under the optimal conditions, TABPT-SPDA-COF shows outstanding enrichment capacity for PPTs in contrast to commercial absorbents ascribed to size selectivity and multiple interaction effects. The method exhibits excellent linear range (0.005-100 ng mL -1 ), satisfactory limits of detection (0.1 pg mL -1 ) as well as relative recoveries (86.2-116 %). This work offers a practicable platform to monitor trace PPTs from complex animal-derived foodstuffs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Since the outbreak of coronavirus disease 2019, the global demand for vaccines has increased rapidly to prevent infection and protect high-risk populations. However, identifying viral mutations poses an additional challenge for chromatographic purification of vaccines and subunit vaccines. In this study, a new affinity peptide model, X 1 VX 2 GLNX 3 WX 4 RYSK, was established, and a library of 612 peptides was generated for ligand screening. Based on a multistep strategy of ligand screening, 18 candidate peptides were obtained. The top ranking peptide, LP14 (YVYGLNIWLRYSK), and two other representative peptides, LP02 and LP06, with lower rankings were compared via molecular dynamics simulation. The results revealed that peptide binding to the receptor binding domain (RBD) was driven by hydrophobic interactions and the key residues involved in the binding were identified. Surface plasmon resonance analysis further confirmed that LP14 had the highest affinity for the wild RBD (K d =0.520 μmol/L), and viral mutation had little influence on the affinity of LP14, demonstrating its great potential as a broad-spectrum ligand for RBD purification. Finally, chromatographic performance of LP14-coupled gel-packed column verified that both wild and omicron RBDs could be purified and were eluted by 0.1 mol/L Gly-HCl buffer (pH 3.0). This research identified a broad-spectrum peptide for RBD purification based on rational design and demonstrated its potential application in the purification of RBDs from complex feedstock., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The linkages of disulfide bond (DSB) play important roles in protein stability and activity. Mass spectrometry-based (MS-based) techniques become accepted tools for DSB analysis in the recent decade. In the bottom-up approach, after enzyme digestion, the neighbouring amino acids of cysteines have great impacts on the physicochemical properties of resulting disulfide bond peptides, determining their retention behaviour on liquid chromatography (LC) and their MS ionization efficiency. In this study, the addition of supercharging reagent in LC mobile phase was used to examine the impact of supercharging reagent on the charge states of disulfide-bond peptides. The results showed that 0.1 % m-nitrobenzyl alcohol (m-NBA) in LC mobile phase increased the sensitivity and charge states of DSB peptides from our model protein, equine Interleukin-5 (eIL5), as well as the resolution of reversed-phase chromatography. Notably, also the sensitivity of C-terminal peptide with His-tag significantly improved. Our findings highlight the effectiveness of employing m-NBA as a supercharging reagent when investigating disulfide-linked peptides and the C-terminal peptide with a His-tag through nano-liquid chromatography mass spectrometry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

In this work, two mixed-mode columns from a different manufacturers and one marketed as a reversed-phase column were characterized and compared in the terms of their interaction abilities, retentivity, peak symmetry, and applicability for peptide separation. All the tested columns contain octadecyl ligand and positively charged modifier, i.e. pyridyl group for the reversed-phase column XSelect CSH C18, quaternary alkylamine for mixed-mode column Atlantis PREMIER BEH C18 AX, and permanently charged moiety (details not available from the manufacturer) for mixed-mode column Luna Omega PS C18. For detailed characterization and comparison of their interaction potential, several approaches were used. First, a simple Walters test was performed to estimate hydrophobic and silanophilic interactions of the tested columns. The highest values of both parameters were observed for column Atlantis PREMIER BEH C18 AX. To investigate the effect of pH and buffer concentration on retention, mobile phases composed of acetonitrile and buffer (ammonium formate, pH 3.0; ammonium acetate pH 4.7 and pH 6.9) in various concentrations (5mM; 10mM; 15mM and 20mM) were used. The analysis of permanently charged compounds was used to describe the electrostatic interaction abilities of the stationary phases. The most significant contribution of electrostatic interactions to the retention was observed for Atlantis PREMIER BEH C18 AX column in the mobile phase with buffer of pH 3.0. A set of ten dipeptides, three pentapeptides and one octapeptide was used to investigate the effects of pH and buffer concentration on retention and peak symmetry. Each of the tested columns provides the optimal peak shape under different buffer pH and concentration. The gradient separation of the 14 tested peptides was used to verify the application potential of the tested columns for peptide separation. The best separation was achieved within 4 minutes on column Atlantis PREMIER BEH C18 AX., Competing Interests: Declaration of Competing Interest The authors declared no conflict of interest., (Copyright © 2021. Published by Elsevier B.V.)

The differentiation of mobile phase compositions between sub-classes which exhibit distinct chromatographic selectivity (i.e. termed characterisation) towards a range of peptide probes with diverse functionality and hence the possibility for multi-modal retention mechanisms has been undertaken. Due to the complexity of peptide retention mechanisms in given mobile phase conditions, no attempt has been made to explain these, instead mobile phases have simply been classified into distinct groups with an aim of identifying those yielding differing selectivities for use in strategic method development roadmaps for the analysis of peptide mixtures. The selectivity differences between nine synthetic peptides (fragments of [Ile27]-Bovine GLP-2) were used to assess how fifty-one RPC mobile phase compositions of differing pH (range 1.8 - 7.8), salt types, ionic strengths, ion-pair reagents and chaotropic / kosmotropic additives affected chromatographic selectivity on a new generation C18 stationary phase (Ascentis Express C18). The mobile phase compositions consisted of commonly used and novel UV or MS compatible additives. The chemometric tool of Principal Component Analysis (PCA) was used to visualise the differences in selectivity generated between the various mobile phases evaluated. The results highlight the importance of screening numerous mobile phases of differing pH, ion-pair reagents and ionic strength in order to maximise the probability of achieving separation of all the peptides of interest within a complex mixture. PCA permitted a ranking of the relative importance of the various mobile phase parameters evaluated. The concept of using this approach was proven in the analysis of a sample of Bovine GLP-2 (1-15) containing synthesis related impurities. Mobile phases with high ionic strength were demonstrated to be crucial for the generation of symmetrical peaks. The observations made on the C18 phase were compared on three additional stationary phases (i.e. alkyl amide, fluorophenyl and biphenyl), which had previously been shown to possess large selectivity differences towards these peptides, on a limited sub-set of mobile phases. With the exception of the ion-pair reagent, similar trends were obtained for the C18, fluorophenyl and biphenyl phases intimating the applicability of these findings to the vast majority of RPC columns (i.e. neutral or weakly polar in character) which are suitable for the analysis of peptides. The conclusions were not relevant for columns with a more disparate nature (i.e. containing a high degree of positive charge)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Screening and monitoring endogenous peptides from complicated biosamples is still a major challenge in mass spectrometry-based proteomics research, mainly due to their low concentration and the interference of high-abundance proteins and other contaminants in biological samples. Herein, a facile and novel approach was described for rapid fabrication of C 18 -functionalized magnetic nanocomposites (C 18 -MNCs) based on one-step aqueous coating of C 18 -Val-Lys-Val-Lys-Val-Lys (C 18 -VK-VI) for the highly selective enrichment of low-abundance endogenous peptides from biological samples. C 18 -VK-VI can readily self-assemble into complete monolayers mainly composed of β-sheets with C 18 hydrophobic chains erecting on the surface of GO@Fe 3 O 4 MNCs under the physiological conditions. The resulting C 18 VK-VI-GO@Fe 3 O 4 MNCs exhibited good performance for peptides enrichment from digests of standard protein (myoglobin, MYO) and human serum, such as high sensitivity (0.05 fmol μL -1 ) and selectivity (mass ratio of MYO digests and MYO = 1:500), rapid separation, and good reproducibility. Such a simple mild and rapid one-step aqueous coating method on the basis of oligopeptides self-assembly showed great potential in surface functionalization of various nanoadsorbents for proteome/peptidome researches., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Following the consolidation of therapeutic proteins in the fight against cancer, autoimmune, and neurodegenerative diseases, recent advancements in biochemistry and biotechnology have introduced a host of next-generation biotherapeutics, such as CRISPR-Cas nucleases, stem and car-T cells, and viral vectors for gene therapy. With these drugs entering the clinical pipeline, a new challenge lies ahead: how to manufacture large quantities of high-purity biotherapeutics that meet the growing demand by clinics and biotech companies worldwide. The protein ligands employed by the industry are inadequate to confront this challenge: while featuring high binding affinity and selectivity, these ligands require laborious engineering and expensive manufacturing, are prone to biochemical degradation, and pose safety concerns related to their bacterial origin. Peptides and pseudopeptides make excellent candidates to form a new cohort of ligands for the purification of next-generation biotherapeutics. Peptide-based ligands feature excellent target biorecognition, low or no toxicity and immunogenicity, and can be manufactured affordably at large scale. This work presents a comprehensive and systematic review of the literature on peptide-based ligands and their use in the affinity purification of established and upcoming biological drugs. A comparative analysis is first presented on peptide engineering principles, the development of ligands targeting different biomolecular targets, and the promises and challenges connected to the industrial implementation of peptide ligands. The reviewed literature is organized in (i) conventional (α-)peptides targeting antibodies and other therapeutic proteins, gene therapy products, and therapeutic cells; (ii) cyclic peptides and pseudo-peptides for protein purification and capture of viral and bacterial pathogens; and (iii) the forefront of peptide mimetics, such as β-/γ-peptides, peptoids, foldamers, and stimuli-responsive peptides for advanced processing of biologics., Competing Interests: Declaration of Competing Interest No personal or financial interests exist., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Chromatographic parameters have been evaluated regarding the supercritical fluid chromatography of amino acids as model compounds for polar and chargeable substances of biochemical and biopharmaceutical interest. Their inherent hydrophilicity requires suitable chromatographic conditions regarding stationary and mobile phase to obtain suitable solubility and peak performance. Ten stationary phases differing in polarity and type of ligands including neutral and charged ones were investigated regarding selectivity, peak performance and complementarity/orthogonality. The mobile phase composition regarding solvent and type of buffer additives by varying the degree of hydrophobicity of the acids and bases and degree of substitution in case of the basic amines. Addition of water to the methanol solvent was furthermore found crucial for obtaining a high peak performance. Some complementary selectivity could be obtained depending on a choice of column. Temperature and pressure variations basically only influenced retention with no selectivity changes. Suitable conditions obtained for the amino acids were then successfully applied to oligo- and polypeptides enabling a separation of 4 polypeptides and differing in only one of the 39 amino acids, thereby mimicking analysis of biotransformation reaction products. The utilization of water as mobile phase additive and sulfonic acids as buffer agents could serve as a generic methodology for polar and charged compounds such as amino containing biomolecules., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

A twin-column Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) process has been developed for the purification of a therapeutic peptide, glucagon, from a crude synthetic mixture. This semi-continuous process uses two identical columns operating either in interconnected or in batch mode, thus enabling the internal recycle of the portions of the eluting stream which do not comply with purity specifications. Because of this feature, which actually results in the simulated countercurrent movement of the stationary phase with respect to the mobile one, the yield-purity trade-off typical of traditional batch preparative chromatography can be alleviated. Moreover, the purification process can be completely automatized. Aim of this work is to present a simple procedure for the development of the MCSGP process based on a single batch experiment, in the case of a therapeutic peptide of industrial relevance. This allowed to recover roughly 90% of the injected glucagon in a purified pool with a purity of about 90%. A comparison between the performance of the MCSGP process and the classical single column batch process indicates that percentage increase in the recovery of target product is +23% when transferring the method from batch conditions to MCSGP, with an unchanged purity of around 89%. This improvement comes at the expenses of a reduction of about 38% in productivity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Affinity chromatography is generally regarded as a powerful tool allowing the single step purification of recombinant proteins with high purity and yields. However, for most protein products, affinity purification methods for industrial applications are not readily available, mainly due to the lack of specific and robust natural counterparts that could function as affinity ligands. In this study, we explored the applicability of nanobody-based peptide-tag immunorecognition systems as a platform for affinity chromatography. Two typical nanobodies (BC2-nb and Syn2-nb) that are capable of recognizing specifically a particular peptide-tag, were prepared through prokaryotic expression and proved to be able to bind with nanomolar affinity to their cognate tag fused to enhanced green fluorescent protein (eGFP). Through an epoxy-based immobilization reaction, the two nanobodies were coupled on a Sepharose CL-6B matrix under the same conditions. The remaining antigen binding activity of the immobilized BC2-nb and Syn2-nb was determined to be 83.1% and 42.9%, yielding the resins with the dynamic binding capacity (DBC) of 21.4 mg/mL and 5.9 mg/mL, respectively. The immobilized affinity ligands exhibited high binding specificity towards their respective target peptides, yielding a product purity above 90% directly from crude bacterial lysates in one single chromatographic step. However, for the both affinity complexes, desorption has been found difficult, and effective recovery of the bound products could be only achieved with competitive elution or after employing harsh conditions such as 10 mM NaOH solution, which will compromise the reuse cycles of the affinity resins. This study shows the potential of nanobody-based affinity chromatography for efficient purification of recombinant proteins especially from complex feedstocks and reveals the primary issues to be addressed to develop a successful application., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

The chiral separations of small peptides is an important challenge in the biological and medical sciences, because different stereoisomers of chiral drugs can often possess different pharmacological, pharmacokinetic, and/or toxicological activities. Commercially available crown ether chiral stationary phases based on S-(3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6 (CROWNPAK CR-I (+)) and (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (ChiroSil RCA (+)) have been successfully used for separating enantiomers of various racemic compounds containing primary amino groups. In this investigation, enantioresolution of more complex model analyte - tetrapeptide Tyr-Arg-Phe-Lys-NH 2 , has been reported on crown ether chiral stationary phases. Organic and acidic modifier content in aqueous mobile phase was tested. All Tyr-Arg-Phe-Lys-NH 2 stereoisomers showed U-shaped retention plots, based on ACN content in mobile phase. Increased retention of tetrapeptide stereoisomers was observed at low (<35%) and at high (>70%) acetonitrile content in the mobile phase, indicating that different separation mechanisms are most likely involved. As a result, baseline separation of all eight tetrapeptide enantiomer pairs was achieved under isocratic elution mode on both chiral columns., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The Peptide RPC Column Characterisation Protocol was applied to 38 stationary phases, varying in ligand chemistry, base silica, end capping and pore size, which are suitable for the analysis of peptides. The protocol at low and intermediate pH is based on measuring retention time differences between peptides of different functionality to calculate selectivity delta values. The characterisation was designed to explore increases / decreases in positive or negative charge (deamidation), steric effect (i.e. racemisation / switch in amino acid order), oxidation and addition / removal of aromatic moieties. The necessity of developing a characterisation protocol specifically for peptide analysis was highlighted by the fact that the small molecule databases (Snyder's Hydrophobic Subtraction Model and the extended Tanaka protocol) failed to correlate with the Peptide RPC Column Characterisation Protocol. Principal Component Analysis was used to demonstrate that the protocol could be used to identify columns with similar or dissimilar chromatographic selectivity for the purpose of selectivity back-up or method development columns respectively. This was validated using peptide fragments derived from the tryptic digest of bovine insulin and carbonic anhydrase. It was also demonstrated that the presence of positively charged functional groups on the stationary phase was advantageous as it yielded very different chromatographic selectivity and improved peak shape., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Capillary electrophoresis tandem mass spectrometry (CE-MS/MS) is an interesting tool for proteomic analysis as the separation principle is orthogonal to liquid chromatography tandem mass spectrometry (LC-MS/MS). The combination of both techniques can bring complementary information to enlarge proteome coverage. In this study, sample preconcentration techniques were investigated in order to improve sample loading and therefore sensitivity. Dynamic pH junction (DPJ) was found to be the most interesting approach by using 200 mM ammonium acetate (NH 4 Ac) adjusted to pH 10.0 as sample matrix. The use of DPJ allowed the identification of more peptides and proteins compared to conventional injections. Moreover, the sheath liquid (SL) composition was optimized in order to enhance signal intensity. A nanoflow SL interface (EMASS-II) was compared to the traditional coaxial SL interface (Triple tube) in terms of number of identified and proteins as well as detection sensitivity (peak area and peak height). MS acquisition was performed using both data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes. The results showed that the combined use of these two acquisition modes provided additional information in terms of identification. Moreover, the use of EMASS-II interface allowed the identification of approximately two times more peptides and proteins. Besides, there was an improvement in sensitivity using EMASS-II as peak height and peak area were improved by 4 and 6-fold, respectively, compared to the Triple tube. Altogether, by combining an efficient sample preconcentration method, a nanoflow CE-MS interface and a hybrid ion-mobility qTOF mass spectrometer, a satisfying sequence coverage was obtained by analyzing 1 µg of E. coli proteome digest., Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

In the present work, we have investigated the combination of hydrophilic interaction liquid chromatography (HILIC) and reversed phase liquid chromatography (RPLC) for the separation of peptides in on-line HILIC x RPLC. This combination usually leads to significant solvent strength mismatch, since a weak solvent in HILIC becomes a strong solvent in RPLC. This may result in band broadening, peak distortion, and breakthrough phenomena. Our focus was directed towards the reduction of band broadening and peak distortion. The conditions of the emergence of breakthrough could be investigated with high resolution mass spectrometry (HRMS) detection. The importance of both the injection volume and the difference in composition between injection and elution solvents was highlighted. Reported strategies to avoid bad peak shapes mostly rely either on flow splitting to limit the injection volume, or on on-line dilution. Here, we propose an alternative approach which consists in injecting large volumes in the second dimension. In this case, no flow-splitting nor dilution prior to the second dimension is required. Our results show that above a certain critical injected volume, depending on both the compound and the elution conditions, narrow and symmetrical peaks can be obtained, despite the persistence of breakthrough. As a result, the injected volume in the second dimension must be larger than the largest critical volume. This counter-intuitive approach was applied for the on-line HILIC x RPLC-UV-HRMS analysis of a complex tryptic digest sample. A peak capacity close to 1500 could be achieved in 30 min, which is two-fold higher than in RPLC x RPLC within the same analysis time., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The development of multifarious stationary phases is still a growing demand so as to solve the tasks of ever evolving actual applications. Herein, with D-2-allylglycine hydrochloride (AG·HCl) as the hydrophilic monomer, diene ionic liquid 1-allyl-3-vinylimidazolium bromide (AVI·Br) and polyhedral oligomeric silsesquioxane methacryl substituted (POSS-MA) as the dual crosslinkers, the highly cross-linked imidazolium-bridged POSS-AVI-AG hybrid monolithic column was fabricated via the "one-pot" free radical copolymerization. The AG·HCl embedded POSS-AVI-AG column displays typical reversed-phase liquid chromatography/hydrophilic interaction liquid chromatography mixed-mode retention mechanisms. Both hydrophobic phenols, alkylbenzenes, aromatic amines and hydrophilic nucleosides/nucleic acid bases, amides and thioureas were successfully separated with high column efficiencies (up to 571,000 plates/m for amides), outperforming our previously reported AVI·Br modified POSS-AVI column. Moreover, the column was also explored for the separation of cytochrome c tryptic digests and egg white protein extraction. All these results demonstrate that the POSS-AVI-AG column has a good potential in separation of both small molecules and complex biological samples with multiple mechanisms., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The robustness of the Peptide Reversed Phase Chromatography (RPC) Column Characterisation Protocol was evaluated using reduced factorial design, to ascertain the degree of control required for parameters including temperature, flow rate, dwell volume, a systematic shift in the gradient, amount of formic acid in the aqueous and organic, pH of the ammonium formate and amount of acetonitrile (%MeCN) in the strong solvent, where a loss of MeCN resulted in an unacceptable variation. Mitigations have been introduced to ensure the integrity of the data to allow RPC columns to be characterised using peptides as probes, with the definitive protocol described. In addition, the instrument and column batch to batch variability were assessed with good reproducibility., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A protocol was defined which utilised peptides as probes for the characterisation of reversed phase chromatography peptide separation systems. These peptide probes successfully distinguished between differing stationary phases through the probe's hydrophobic, electrostatic, hydrogen bonding and aromatic interactions with the stationary phase, in addition, to more subtle interactions such as the phase's ability to separate racemic or isomeric probes. The dominating forces responsible for the chromatographic selectivity of peptides appear to be hydrophobic as well as electrostatic and polar in nature. This highlights the need for other types of stationary phase ligands with possibly mixed mode functionalities / electrostatic / polar interactions for peptide separations rather than the hydrophobic ligands which dominate small molecule separations. Selectivity differences are observed between phases, but it appears that it is the accessibility differences between these phases which play a crucial role in peptide separations i.e. accessibility to silanols, the hydrophobic acetonitrile / ligand layer or a thin adsorbed water layer on the silica surface., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Deamidation of asparagine (Asn) residues of monoclonal antibodies (mAbs) plays a pivotal role in the in vivo/vitro degradation or efficacy loss of biopharmaceuticals. However, a major challenge for MS analysis of deamidation of Asn-containing peptides in mAbs, is due to the fact that there is only a 1 Da mass shift between the native form (Asn residues) and deamidated forms (n-aspartyl (n-Asp) and isoaspartyl (isoAsp) residues with identical mass). Therefore, a chromatographic separation of the deamidated proteins and/or the peptides derived therefrom is needed prior to MS analysis. In this study, the monolithic column with various stationary phases, including reverse phase (RP), single phospholipid-functionalized and mixed phospholipid-functionalized monoliths, were prepared for the separation of the deamidation-sensitive signature peptide (IYPTNGYTR) of trastuzumab and its two deamidated products, n-Asp55 residue IYPTDGYTR and isoAsp55 residue IYPTisoDGYTR. Compared to the RP monolith, the phospholipid-functionalized monoliths provided mixed-mode interactions and exhibited better peak shape and separation selectivity. The effect of the parameters, including the type and concentration of buffer, temperature and pH value on the separation performance were investigated. Under the optimal conditions, the three peptides were fully separated on a mixed phosphocholine (PC) / phosphatidic acid (PA) functionalized monolith (poly (MDPC 60 PA 40 -co-EDMA)) due to the joint contribution of hydrophobic and electrostatic interactions. Therefore, the novel method based on the mixed phospholipids-functionalized monolithic column exhibited good potential for the analysis of deamidated peptides, which will be useful for the in-depth study of post-translational modifications of mAbs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Reversed-phase chromatography is the most common technique for separation of tryptic peptides. In this short communication, we describe the optimization of sample loading and separation parameters for a novel micromachined column and provide a detailed description on the performance and reproducibility of this separation system. Tryptic digest of a mixture of seven proteins with diverse mass and isoelectric point was used as a test sample. The methods developed and used are straight-forward; by using well-balanced, combined-step gradients an optimal distribution of peptides on the column could be achieved throughout the complete gradient window. The potential use of the column is exceptional due to the low back-pressure, better distribution of peptides over the separation window, enhanced stability and reproducibility of retention times, and the prolonged lifetime of columns compared to conventionally packed nano-HPLC column. The higher identification rates have been demonstrated through measurements of HeLa cell lysates under identical chromatographic conditions on the pillar array and packed-bed columns., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This study is concerned with a chromatography-based approach (Immobilized Metal Ion Affinity Chromatography) for the recovery of gallium binding peptide sequences from a recombinant phage display library. The here described methods apply the fundamental knowledge and methods of separation science and meet thereby the key requirement of the phage display technique of precise separation of target-binding bacteriophage clones from non-interacting bacteriophage during the biopanning. During the chromatopanning called process, a total of 101 bacteriophage clones were identified of which in subsequent binding experiments, phage clones expressing the peptide sequences TMHHAAIAHPPH, SQALSTSRQDLR and HTQHIQSDDHLA were characterized to bind >10 fold better to a target that presents immobilized gallium ions than control phage, displaying no peptide sequence. The performance of biopanning experiments in chromatographic systems is particularly suitable for demanding targets such as trivalent metal ions. We found, that the selection process benefits immensely from the stable immobilization of the target metal ions during the entire biopanning process as well as the complete recovery of well interacting bacteriophage clones. Among others, this was possible due to an enhanced monitoring of process conditions and fractionation of eluates., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A strategy for determining a suitable solvent gradient in silico in preparative peptide separations is presented. The strategy utilizes a machine-learning-based method, called ELUDE, for peptide retention time predictions based on the amino acid sequences of the peptides. A suitable gradient is calculated according to linear solvent strength theory by predicting the retention times of the peptides being purified at three different gradient slopes. The advantage of this strategy is that fewer experiments are needed to develop a purification method, making it useful for labs conducting many separations but with limited resources for method development. The preparative separation of met-enkephalin and leu-enkephalin was used as model solutes on two stationary phases: XBridge C18 and CSH C18. The ELUDE algorithm contains a support vector regression and is pre-trained, meaning that only 10-50 peptides are needed to calibrate a model for a certain stationary phase and gradient. The calibration is done once and the model can then be used for new peptides similar in size to those in the calibration set. We found that the accuracy of the retention time predictions is good enough to usefully estimate a suitable gradient and that it was possible to compare the selectivity on different stationary phases in silico. The absolute relative errors in retention time for the predicted gradients were 4.2% and 3.7% for met-enkephalin and leu-enkephalin, respectively, on the XBridge C18 column and 2.0% and 2.8% on the CSH C18 column. The predicted retention times were also used as initial values for adsorption isotherm parameter determination, facilitating the numerical calculation of overloaded elution profiles. Changing the trifluoroacetic acid (TFA) concentration from 0.05% to 0.15% in the eluent did not seriously affect the error in the retention time predictions for the XBridge C18 column, an increase of 1.0 min (in retention factor, 1.3). For the CSH C18 column the error was, on average, 2.6 times larger. This indicates that the model needs to be recalibrated when changing the TFA concentration for the CSH column. Studying possible scale-up complications from UHPLC to HPLC such as pressure, viscous heating (i.e., temperature gradients), and stationary-phase properties (e.g., packing heterogeneity and surface chemistry) revealed that all these factors were minor to negligible. The pressure effect had the largest effect on the retention, but increased retention by only 3%. In the presented case, method development can therefore proceed using UHPLC and then be robustly transferred to HPLC., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This work proposes a method to separate proteins and polyphenols in a food byproduct with high bioactive properties and demonstrate by high performance liquid chromatography tandem mass spectrometry its efficiency. Bioactive substances were extracted using high intensity focused ultrasounds. Resulting extract (SR) was submitted to a step for the purification of proteins and to obtain a protein isolate (PI). Both extracts (SR and PI) were digested using two different enzymes (alcalase and thermolysin). Antioxidant, hypocholesterolemic, and antihypertensive properties of hydrolysates were explored. High antioxidant capacity, mainly due to the presence of polyphenols, was observed in SR and its hydrolysates. Hydrolysates obtained from PI using the alcalase enzyme showed simultaneously a high capacity to inhibit cholesterol esterase and to reduce micellar cholesterol solubility. Hydrolysate obtained from PI using the thermolysin enzyme showed a high antihypertensive capacity. Peptides and polyphenols in hydrolysates were identified by RP-HPLC-ESI-Q-TOF. Hydrolysates obtained from PI showed a high amount of peptides and a negligible amount of polyphenols while polyphenols were mainly present in hydrolysates from SR., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Peptide retention standards are widely used by chromatography specialists. They can be used for quality control of peptide separations (separation efficiency, selectivity, retention values) and for accurate concatenation of retention data from multiple acquisitions in proteomics. So far the repertoire of available retention standards is mostly limited to reversed-phase separations. We introduce a synthetic peptide mixture which can be used in conjunction with the most popular peptide separation techniques: reversed-phase (RPLC), strong-cation exchange (SCX), (strong-anion exchange) SAX and hydrophilic interaction liquid chromatography (HILIC). Target sequences were first designed in-silico using Sequence-Specific Retention Calculator models covering all major peptide separation mechanisms. Peptides were also designed while keeping in mind the simplicity of retention time assignment using MS detection: they all have nearly identical masses and identical intense y 3 fragment ions. This contribution demonstrates the application of this mixture for characterization of eight HILIC as well as SAX, SCX and C18 columns., (Copyright © 2018. Published by Elsevier B.V.)

Capillary column packed with silica colloidal crystals (SCC) exhibits great efficiency in liquid chromatography because of its highly-ordered structure and extremely narrow channels (about hundred nanometer in diameter) between the particles. However, problems arise due to the extremely high back-pressure created with its dense packings. It is also a challenge to prepare a stable SCC column with an appropriate length for chromatographic separation on a commercial instrument. We have synthesized monodispersed, sub-micron SiO 2 particles bonded with C18 functionality and fabricated SCC capillary columns up to 100 mm in length. The packing materials in the column displayed Bragg diffraction with blueish color under irradiation with white light, indicating the formation of SCC. The column performance was evaluated using pressurized capillary electrochromatography (pCEC) and column efficiencies of more than a hundred thousand plates per column (plate height < 1 μm) were achieved using peptide as a sample. The run-to-run reproducibility expressed with RSD in terms of retention time and peak area for aromatic compounds were less than 0.076% and 1.96%, respectively. These results demonstrate that the combination of pCEC and SCC columns may provide an innovative analytical technique with extraordinary efficiency, resolution and speed for the separation of complex samples., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We present capillary electrophoresis-mass spectrometry (CE-MS) in combination with advanced chemometric tools for the analysis of bioactive compounds in food, in particular for the identification of antihypertensive peptides in a nutraceutical derived from a bovine milk protein hydrolysate. Different extracts of the nutraceutical were analyzed by CE-MS, and the electropherograms were processed using a novel data analysis workflow that included regions of interest (ROIs) compression and multivariate curve resolution alternating least squares (MCR-ALS). MCR-ALS permitted the description of the nutraceutical extract as ten characteristic components with their electrophoretic profiles and mass spectra. Twenty-two compounds were tentatively identified as antihypertensive bovine casein fragments through a mass search in a database of bioactive peptides. The identity of 17 antihypertensive peptides was reliably confirmed by capillary electrophoresis-tandem mass spectrometry. The proposed analytical approach demonstrated the potential to obtain a characteristic and activity-related fingerprint for quality control and authentication of the antihypertensive nutraceutical., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We are currently examining the potential of amphipathic cationic α-helical peptides as a new generation of peptide standards for both cation-exchange high-performance liquid chromatography and reversed-phase chromatography. Thus, amphipathic peptides are particularly suitable for high-performance liquid chromatography standards due to the preferred binding of the non-polar face to the hydrophobic stationary phase of reversed-phase packings or the preferred binding of the polar face to the charged/hydrophilic stationary phase of cation-exchange packings. The ability of different reversed-phase or cation-exchange matrices to separate mixtures of peptide standards with only subtle hydrophilicity/hydrophobicity variations in both the non-polar and polar face of the peptides can then be assessed. Currently, we have designed de novo a mixture of six 26-residue all D-conformation amphipathic cationic α-helical peptides with a single, positively charged lysine residue in the center of the non-polar face and an increasing number of lysine residues (4-9 residues) replacing neutral residues in the polar face, resulting in an overall net positive charge of +5 to +10. Thus, the non-polar, preferred reversed-phase chromatography binding face remains constant, with only the polar face varying in hydrophilicity/hydrophobicity. Interestingly, even with the non-polar face remaining constant, reversed-phase columns of varying functional group properties (e.g., C 8 , C 18 , phenyl, polar endcapped, polar embedded) and porosity (porous versus superficially porous) were able to separate the six peptides in aq. TFA/acetonitrile gradients, albeit with different selectivities. The value of the standards in cation-exchange chromatography was expressed by monitoring the requirement of acetonitrile (0-40% in the mobile phase) to overcome hydrophobic interactions of the peptides with the cation-exchange matrix matrix when eluting with sodium perchlorate gradients at pH 6.5. Interestingly, the resolution of the higher charged peptides (+8,+9,+10) was particularly sensitive to acetonitrile levels. Our results clearly demonstrate the excellent potential of these novel peptide standards to enable optimal column choice and mobile phase conditions for reversed-phase chromatography and cation-exchange chromatography for peptide separations., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We investigated and compared the robustness of supercritical fluid chromatography (SFC) separations of the peptide gramicidin, using either isocratic or gradient elution. This was done using design of experiments in a design space of co-solvent fraction, water mass fraction in co-solvent, pressure, and temperature. The density of the eluent (CO 2 -MeOH-H 2 O) was experimentally determined using a Coriolis mass flow meter to calculate the volumetric flow rate required by the design. For both retention models, the most important factor was the total co-solvent fraction and water mass fraction in co-solvent. Comparing the elution modes, we found that gradient elution was more than three times more robust than isocratic elution. We also observed a relationship between the sensitivity to changes and the gradient steepness and used this to draw general conclusions beyond the studied experimental system. To test the robustness in a practical context, both the isocratic and gradient separations were transferred to another laboratory. The gradient elution was highly reproducible between laboratories, whereas the isocratic system was not. Using measurements of the actual operational conditions (not the set system conditions), the isocratic deviation was quantitatively explained using the retention model. The findings indicate the benefits of using gradient elution in SFC as well as the importance of measuring the actual operational conditions to be able to explain observed differences between laboratories when conducting method transfer., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A microfluidic glass chip for performing sensitive mass spectrometry (MS) is developed by integrating nanochannel-based electropreconcentration with electrospray ionization (ESI). Cation analytes in acidic buffer conditions (∼pH 3) were preconcentrated at a micro-nano-microchannel junction where a positively charged polymer, polyE-323, is coated to reverse the electroosmotic flows (anodic EOF) and prevent sample adsorption. The preconcentrated cation analytes were delivered to a monolithic spray tip using an integrated field-free electrokinetic pump, enabling a stable positive ESI with negligible dead volume. The sample preconcentration-ESI-MS achieved one order of magnitude signal enhancement in MS signal with a peptide mixture., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Monodispersed core-shell silica spheres with fibrous shell structure and tunable pore size were prepared by using a one-pot oil-water biphase method. The pore size could be tuned from 7 nm to 37 nm by using organic solvents with different polarities as oil phase. The spheres synthesized by using benzene as organic solvent had the maximum pore size of 37 nm and possessed a surface area of 61 m 2  g -1 . The obtained wide pore core-shell silica spheres were applied for rapidly separating small molecules, peptides, small proteins, and large proteins with molecular weight up to 200 kDa. Since the pore size of the core-shell silica spheres was sufficiently large for the free access of all the solutes, sharp and symmetric peaks were obtained. The separation performance was as high as 264,531 plates m -1 for fluorene. The great efficient separation demonstrates that the wide pore core-shell silica spheres have a great potential for rapid analysis of both small and large solutes with high performance liquid chromatography., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In our paper we investigate the influence of pH of the mobile phase buffer, on retention of peptides in both: normal phase (NP) and reversed phase (RP) high-performance thin-layer chromatography systems (HPTLC), using C18 silica-based adsorbent. We also compare the influence of pH on retention and/or migration distance of peptides in separation systems of HPTLC and PPEC (pressurized planar electrochromatography) techniques. Our results show, that the change of pH of the mobile phase buffer can be used for two-dimensional HPTLC separation of peptides, but much stronger change of separation selectivity can be obtained by overall inversion of separation system type (NP/RP), as we have described before (Gwarda et al., 2016). In PPEC influence of the mobile phase pH on selectivity of peptide separation is very clear and strictly different than that in HPTLC (including overall inversion of direction of solutes migration), what results from the significant share of electrophoretic effect in overall mechanism of separation. Migration of peptides in two opposite directions results in better selectivity of separation by PPEC. In separation systems of both HPTLC and PPEC techniques, the change of pH clearly affects the efficiency of the separation system, so it can be used for optimization of separation conditions. Our results also show, that some modifications of PPEC equipment and materials could be useful for application of this technique in the field of peptide analysis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Peptide separation selectivity of four different HILIC sorbents was compared through the development of sequence-specific retention prediction algorithms for the datasets of 36-40 thousands peptides each. Hydrophilicity of these sorbents at pH 4.5 (peptide retention under acetonitrile:water gradients) increases in the following order: Luna HILIC < XBridge Amide < Atlantis Silica ∼ Luna HILIC Silica. Bare silica phases are characterized by higher retention coefficients for basic residues (Arg, Lys, His), while interactions with neutral HILIC phases is driven by interaction with the charged residues (Asp, Glu, Arg, Lys, His). Such difference is caused by electrostatic repulsion of negatively charged side chains of Asp and Glu from silanol groups on bare silica sorbents. The sequence-specific features characteristic for both bonded and bare-silica supports were established. Variation of retention coefficients at N- and C-termini showed a peptide orientation effect on silica matrices. The negatively charged surface of silica attracts positively charged groups, thus increasing interaction of N-terminal residues. The effect of peptide helicity is also different between two types of sorbents. The contribution of amphipathic helicity was found to be higher for more hydrophilic silica matrices, causing a reduced predictive accuracy of the models: all four Sequence-Specific Retention Calculator HILIC models had R 2 -values in 0.973-0.98 range. Development of accurate prediction models provides a solid support for retention prediction filtering of false positive identifications in high-throughput proteomics analyses, guided development of 2D LCMS procedures ((HILIC-RP)). Our analysis also shows that all phases possess a high degree of orthogonality to RPLC (formic acid) with Luna HILIC being the best choice in this regard., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Computer-aided method-development programs require accurate models to describe retention and to make predictions based on a limited number of scouting gradients. The performance of five different retention models for hydrophilic-interaction chromatography (HILIC) is assessed for a wide range of analytes. Gradient-elution equations are presented for each model, using Simpson's Rule to approximate the integral in case no exact solution exists. For most compound classes the adsorption model, i.e. a linear relation between the logarithm of the retention factor and the logarithm of the composition, is found to provide the most robust performance. Prediction accuracies depended on analyte class, with peptide retention being predicted least accurately, and on the stationary phase, with better results for a diol column than for an amide column. The two-parameter adsorption model is also attractive, because it can be used with good results using only two scanning gradients. This model is recommended as the first-choice model for describing and predicting HILIC retention data, because of its accuracy and linearity. Other models (linear solvent-strength model, mixed-mode model) should only be considered after validating their applicability in specific cases., (Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.)

A facile approach was developed to prepare hybrid monoliths with different functions via two-step photo-initiated reactions. Firstly, acrylopropyl polyhedral oligomertic silsesquioxane (acryl-POSS) and propargyl acrylate (PA) were used as precursors to synthesize alkynyl-functionalized hybrid monoliths via photo-initiated free radical polymerization. Secondly, the hybrid monoliths were modified with 1-octadecanethiol (ODT) and sodium 3-mercapto-1-propanesulfonate (SMPS) via photo-initiated thiol-yne click reaction to prepare reversed-phase (RP) and strong cation-exchange (SCX) hybrid monoliths, respectively. The results of chromatographic characterization indicated that the column efficiencies for alkylbenzenes on ODT-modified hybrid monolith reached 84,000-87,700 plates per meter at the velocity of 0.58mm/s, and also revealed a retention-independent efficient performance of small molecules in isocratic elution. The SMPS-modified hybrid monolith exhibited both hydrophobicity and ion-exchange mechanisms, and the dynamic binding capacity was calculated to be 1.4×10 -4 μmol/cm. Human Hela cells tryptic digest was well separated on ODT-modified hybrid monolith in one-dimensional RPLC-MS/MS, and 2786 unique peptides and 685 proteins were identified. Furthermore, the SMPS-modified monolith coupled with ODT-modified monolith was used for two-dimensional separation of human Hela cells tryptic digest in SCX-RPLC-MS/MS, and the results showed that 9744 unique peptides and 2749 proteins were identified. Compared to those identified in one-dimensional RP system, the total numbers of unique peptides and proteins identified in SCX-RP system increased by 249.7% and 301.3%, respectively., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Hydrogen deuterium exchange mass spectrometry (HDX MS) reports on the conformational landscape of proteins by monitoring the exchange between backbone amide hydrogen atoms and deuterium in the solvent. To maintain the label for analysis, quench conditions of low temperature and pH are required during the chromatography step performed after protease digestion but before mass spectrometry. Separation at 0°C is often chosen as this is the temperature where the most deuterium can be recovered without freezing of the typical water and acetonitrile mobile phases. Several recent reports of separations at subzero Celsius emphasize the promise for retaining more deuterium and using a much longer chromatographic gradient or direct infusion time. Here we present the construction and validation of a modified Waters nanoACQUITY HDX manager with a third temperature-controlled zone for peptide separations at subzero temperatures. A new Peltier-cooled door replaces the door of a traditional main cooling chamber and the separations and trapping column are routed through the door housing. To prevent freezing, 35% methanol is introduced post online digestion. No new pumps are required and online digestion is performed as in the past. Subzero separations, using conventional HPLC column geometry of 3μ m particles in a 1×50mm column, did not result in major changes to chromatographic efficiency when lowering the temperature from 0 to -20°C. There were significant increases in deuterium recovery for both model peptides and biologically relevant protein systems. Given the higher levels of deuterium recovery, expanded gradient programs can be used to allow for higher chromatographic peak capacity and therefore the analysis of larger and more complex proteins and systems., (Copyright © 2017 Elsevier B.V. All rights reserved.)

While capillary zone electrophoresis (CZE) provides dramatically improved numbers of peptide identifications compared with reversed-phase chromatography for bottom-up proteomics of mass limited samples, CZE inevitably produces lower numbers of peptide identifications than RPLC for larger samples. One reason for this poorer performance is the dead time between injection of samples and subsequent appearance of the fastest moving component. This dead time is typically 25% of the separation window in CZE, but is only 5% of the separation window in gradient elution RPLC. This dead time can be eliminated in CZE by use of a multisegment injection mode where a series of samples is analyzed by injecting each sample while the preceding sample is still being separated. In this paper, we demonstrate that capillary zone electrophoresis employing sequential injections can produce a doubling in peptide identification rate with no degradation in separation efficiency., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Enhanced-fluidity liquid chromatography (EFLC) methods using methanol/H 2 O/CO 2 and hydrophilic interaction liquid chromatography (HILIC) were explored for the separation of proteins and peptides. EFLC is a separation mode that uses a mobile phase made of conventional solvents combined with liquid carbon dioxide (CO 2 ) in subcritical conditions. The addition of liquid CO 2 enhances diffusivity and decreases viscosity while maintaining mixture polarity, which typically results in reduced time of analysis. TFA additive and elevated temperature were leveraged as key factors in the separation of a 13-analyte intact protein mixture in under 5min. Under these conditions EFLC showed modest improvement in terms of peak asymmetry and analysis time over the competing ACN/H 2 O separation. Protein analytes detected by electrospray ionization - quadrupole time of flight, were shown to be unaffected by the addition of CO 2 in the mobile phase. Herein, the feasibility of separating hydrophilic proteins up to 80kDa (with transferrin) is demonstrated for CO 2 -containing mobile phases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Orthogonality metrics (OMs) for three and higher dimensional separations are proposed as extensions of previously developed OMs, which were used to evaluate the zone utilization of two-dimensional (2D) separations. These OMs include correlation coefficients, dimensionality, information theory metrics and convex-hull metrics. In a number of these cases, lower dimensional subspace metrics exist and can be readily calculated. The metrics are used to interpret previously generated experimental data. The experimental datasets are derived from Gilar's peptide data, now modified to be three dimensional (3D), and a comprehensive 3D chromatogram from Moore and Jorgenson. The Moore and Jorgenson chromatogram, which has 25 identifiable 3D volume elements or peaks, displayed good orthogonality values over all dimensions. However, OMs based on discretization of the 3D space changed substantially with changes in binning parameters. This example highlights the importance in higher dimensions of having an abundant number of retention times as data points, especially for methods that use discretization. The Gilar data, which in a previous study produced 21 2D datasets by the pairing of 7 one-dimensional separations, was reinterpreted to produce 35 3D datasets. These datasets show a number of interesting properties, one of which is that geometric and harmonic means of lower dimensional subspace (i.e., 2D) OMs correlate well with the higher dimensional (i.e., 3D) OMs. The space utilization of the Gilar 3D datasets was ranked using OMs, with the retention times of the datasets having the largest and smallest OMs presented as graphs. A discussion concerning the orthogonality of higher dimensional techniques is given with emphasis on molecular diversity in chromatographic separations. In the information theory work, an inconsistency is found in previous studies of orthogonality using the 2D metric often identified as %O. A new choice of metric is proposed, extended to higher dimensions, characterized by mixes of ordered and random retention times, and applied to the experimental datasets. In 2D, the new metric always equals or exceeds the original one. However, results from both the original and new methods are given., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Fusion protein systems are commonly used for expression of small proteins and peptides. An important criterion for a fusion protein system to be useful is the ability to separate the protein of interest from the tag. Additionally, because no protease cleaves fusion proteins with 100% efficiency, the ability to separate the desired peptide from any remaining uncleaved protein is also necessary. This is likely to be the more difficult task as at least a portion of the sequence of the fusion protein is identical to that of the protein of interest. When a high level of purity is required, gradient elution reversed-phase HPLC is frequently used as a final purification step. Shallow gradients are often advantageous for maximizing both the purity and yield of the final product; however, the relationship between relative retention times at shallow gradients and those at steeper gradients typically used for analytical HPLC are not always straightforward. In this work, we report reversed-phase HPLC results for the fusion protein system consisting of the N-terminal domain of ribosomal protein L9 (NTL9) and the 36-residue villin headpiece subdomain (HP36) linked by a recognition sequence for the protease factor Xa. This system represents an excellent example of the difficulties in purification that may arise from this unexpected elution behavior at shallow gradients. Additionally, we report on the sensitivity of this elution behavior to the concentration of the additive trifluoroacetic acid in the mobile phase and present optimized conditions for separating HP36 from the full fusion protein by reversed-phase HPLC using a shallow gradient. Finally, we suggest that these findings are relevant to the purification of other fusion protein systems, for which similar problems may arise, and support this suggestion using insights from the linear solvent strength model of gradient elution liquid chromatography., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The need to improve the peak capacity of liquid chromatography motivates the development of two-dimensional analysis systems. This paper presented a fully automated stop-flow two-dimensional liquid chromatography system with size exclusion chromatography followed by reversed phase liquid chromatography (SEC×RPLC) to efficiently separate peptides. The effects of different stop-flow operational parameters (stop-flow time, peak parking position, number of stop-flow periods and column temperature) on band broadening in the first dimension (1 st D) SEC column were quantitatively evaluated by using commercial small proteins and peptides. Results showed that the effects of peak parking position and the number of stop-flow periods on band broadening were relatively small. Unlike stop-flow analysis of large molecules with a long running time, additional band broadening was evidently observed for small molecule analytes due to the relatively high effective diffusion coefficient (D eff ). Therefore, shorter analysis time and lower 1 st D column temperature were suggested for analyzing small molecules. The stop-flow two-dimensional liquid chromatography (2D-LC) system was further tested on peanut peptides and an evidently improved resolution was observed for both stop-flow heart-cutting and comprehensive 2D-LC analysis (in spite of additional band broadening in SEC). The stop-flow SEC×RPLC, especially heart-cutting analysis with shorter analysis time and higher 1 st D resolution for selected fractions, offers a promising approach for efficient analysis of complex samples., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Over the last 30years, (±)-1-(9-fluorenyl)ethyl chloroformate ((±)-FLEC) was used as a chiral derivatizing agent in various analytical applications involving a wide range of endogenous, pharmaceutical and environmentally relevant molecules. This comprehensive review aims to present all the significant aspects related to the state of the art in FLEC labeling and subsequent chiral separation of the resulting diastereomers using LC, SFC and CE techniques., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Chemical or enzymatic modifications of therapeutic monoclonal antibodies (mAbs) having high risk towards safety and efficacy are defined as critical quality attributes (CQAs). During therapeutic mAbs process development, a variety of analytical techniques have to be used for the thorough characterization and quantitative monitoring of CQAs. This paper describes the development of a rapid analytical platform to assess and rank charge variants of mAbs. The workflow is first based on a cation exchange chromatography (CEX) comparative analysis of intact IgGs versus F(ab)'2 and Fc sub-domains generated by IdeS digestion. This analytical procedure was validated with FDA and EMA approved mAbs. Then, functional assays and peptide mapping can be performed in a second instance. This approach can be used during the early stage of drug research and development to screen lead molecules and select optimized candidates (best clone, best formulation) which could be "easily" developed (OptimAbs)., (Copyright © 2017. Published by Elsevier B.V.)

A new on-line multidimensional system for sequential trapping and individual elution and separation of peptides based on their molecular weight is described. By sequentially using two chemically different trapping columns, a polymethacrylate monolith and a packed C18 one, peptides from complex samples can be on-line trapped and divided into two fractions, containing respectively mainly medium-large peptides and smaller peptides. Then, by means of two switching valves working in parallel, the two fractions were individually separated by reversed phase chromatography. The whole gradient consisted of two subgradients, with the first one dedicated to the separation of smaller peptides and the second one to the separation of larger peptides. Such configuration allowed to identify up to 1476 proteins in a standard E. coli tryptic digest, with improved performance, increased average sequence coverage and reduced single unique peptide identifications compared to a conventional shotgun proteomics configuration comprising only the C18 trapping column and the analytical column., (Copyright © 2017 Elsevier B.V. All rights reserved.)

This study was devoted to the search for conditions leading to highly efficient sub-hour separations of complex peptide samples with the objective of coupling to mass spectrometry. In this context, conditions for one dimensional reversed phase liquid chromatography (1D-RPLC) were optimized on the basis of a kinetic approach while conditions for on-line comprehensive two-dimensional liquid chromatography using reversed phase in both dimensions (on-line RPLCxRPLC) were optimized on the basis of a Pareto-optimal approach. Maximizing the peak capacity while minimizing the dilution factor for different analysis times (down to 5min) were the two objectives under consideration. For gradient times between 5 and 60min, 15cm was found to be the best column length in RPLC with sub-2μm particles under 800bar as system pressure. In RPLCxRPLC, for less than one hour as first dimension gradient time, the sampling rate was found to be a key parameter in addition to conventional parameters including column dimension, particle size, flow-rate and gradient conditions in both dimensions. It was shown that the optimum sampling rate was as low as one fraction per peak for very short gradient times (i.e. below 10min). The quality descriptors obtained under optimized RPLCxRPLC conditions were compared to those obtained under optimized RPLC conditions. Our experimental results for peptides, obtained with state of the art instrumentation, showed that RPLCxRPLC could outperform 1D-RPLC for gradient times longer than 5min. In 60min, the same peak intensity (same dilution) was observed with both techniques but with a 3-fold lower injected amount in RPLCxRPLC. A significant increase of the signal-to-noise ratio mainly due to a strong noise reduction was observed in RPLCxRPLC-MS compared to the one in 1D-RPLC-MS making RPLCxRPLC-MS a promising technique for peptide identification in complex matrices., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Multidimensional peptide fractionation strategies have been approved as the efficient approaches to significantly improve the depth of proteome coverage. In this study, a simple and integrated spintip-based protein digestion and three-dimensional peptide fractionation technology (3D-SISPROT) was developed for the deep proteome profiling from low microgram of proteins as starting materials. All the sample preparation steps, including protein digestion, strong anion exchange (SAX)-based fractionation, and high-pH reversed phase (RP) fractionation were integrated into one pipette tip packed with SAX and C 18 membranes for the first time. The SAX plus C 18 membranes design minimizes the sample loss and ensures high efficient SAX-based digestion. 4275 proteins were identified with 1.4h of MS time when 6μg cell lysates was processed. More importantly, the SAX-based digestion procedure did not influence the SAX-based peptide fractionation efficiency which was done in the same SAX membrane. The 3D-SISPROT was exemplified by the analysis of 30μg of HEK 293T cell lysates with 20.4h of MS time, which resulted in the identification of 8222 proteins including 3215 annotated membrane proteins. Gene Ontology annotations indicated that the 3D-SISPROT was unbiased for the proteins from major cellular components. Taking advantages of the efficient SAX-based and high-pH RP-based fractionation strategies, we expect that the 3D-SISPROT can be applied for the deep proteome profiling with limited starting material., (Copyright © 2017 Elsevier B.V. All rights reserved.)

An organic-silica hybrid monolithic capillary column was fabricated by crosslinking (3-aminopropyl)trimethoxysilane (APTMS) modified mesoporous carbon nanoparticles (AP-MCNs) with tetramethoxysilane (TMOS) and n-butyltrimethoxysilane (C4-TriMOS). Scanning electron microscopy (SEM), X-ray photoelectron spectroscopy, mercury intrusion porosimetry and inverse size-exclusion chromatography characterization proved the successful immobilization of mesoporous carbon nanoparticles (MCNs). The crosslinking of AP-MCNs into the hybrid monolithic matrix has significantly increased the reversed-phase retention of alkylbenzenes and chromatographic performance for small molecules separations in comparison with the neat one without MCNs. The resulting column efficiency of the mesoporous carbon nanoparticle-based butyl-silica hybrid monolithic column (MCN-C4-monolith) was up to ca. 116,600N/m for the capillary liquid chromatography (cLC) separation of butylbenzene. Enhanced performance of proteins separation was achieved on the MCN-C4-monolith in comparison with the butyl-silica hybrid monolithic column without MCN (C4-monolith). The separation of peptides from bovine serum albumin (BSA) digest was carried out on the MCN-C4-monolith by capillary liquid chromatography-tandem mass spectrometry (cLC-MS/MS) with protein sequence coverage of 81.9%, suggesting its potential application in proteomics., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Self-preparation of nano liquid chromatography (nLC) columns has advantages regarding cost and flexibility. For targeted proteomics, we evaluated several approaches for particle-packing nLC columns and manufacturing fritless silica-based monolithic trap columns (50μm inner diameter). Our preferred approach for nLC column preparation was to magnetically stir Accucore core shell particles (C18 stationary phase) in ACN/water (80/20, v/v) suspensions during pressure-driven filling of polymer-fritted standard fused silica capillaries. The columns were ready for use about one hour after preparation had begun. They had comparable peak capacities (peptides) to commercial columns, and satisfactory within/between-column retention time repeatability, suited for targeted proteomics. Packing with commercial capillary housings/nanospray emitters did not improve performance compared to packing with in-house fritted stock fused silica capillary tubing. For trap columns, several recipes for narrow bore silica-based monolithic columns were evaluated, and we found the recipe by Zou et al. (2005) to be reproducible. Compared to the standard C18 trap column for Accucore nLC columns, monolith trap columns (C8 stationary phase) significantly reduced peak widths. The readily prepared in-house columns were used for targeted detection of the enzyme CYP27A1 in cancer cells, which is associated with proliferation and metastasis of breast cancer., (Copyright © 2017 Elsevier B.V. All rights reserved.)