Peptide Mixtures Research Articles

Novel casein antimicrobial peptides for the inhibition of oral pathogenic bacteria

Milk casein is a rich source of antimicrobial peptides (AMPs) and the most common way to produce AMPs is enzymatic hydrolysis in vitro. In this study, active casein antimicrobial peptide (CAMPs) mixtures were generated by optimized proteolytic cleavage of milk casein. These natural-safe CAMPs mixtures exhibited high activity in the inhibition of Streptococcus mutans and Porphyromonas gingivalis. Morphological characterization suggested the pathogenic bacteria presented incomplete or irregular collapsed membrane surface after the treatment with active CAMPs mixtures. The CAMPs inhibition activity was also effective in the attachment and development of microbial biofilm. Potential CAMPs sequences were unambiguously determined by unbiased proteomic analysis and 301 potential CAMPs were obtained. The activity of 4 novel CAMPs was successfully confirmed by using synthetic standards. This study provides a promising milk CAMPs resource for the development of safe agents in oral bacteria inhibition and functional foods.

The Significance of Hypervariability and Conserved Motifs in Antimicrobial Peptides from Tree Frogs.

Amphibians' skin is a rich source of natural antimicrobial peptides (AMPs). These AMPs exhibit marked inter- and intraspecific sequence divergence linked to the arms race between host and pathogens. Here, we combine peptidomics, molecular modeling, and phylogenetic analyses to understand the evolution of AMPs in Cophomantini, a diverse clade of neotropical tree frogs, and to investigate their interaction with bacterial membranes. Consistent with results in other amphibians, all species of Cophomantini secrete a mixture of peptides. We selected the hylin peptide family to survey sequence variability and the presence of common amino acid motifs. We found that most species secrete a unique set of hylins that, though variable, share the conserved motif Gly-X-X-X-Pro-Ala-X-X-Gly, with Gly and Pro colocalizing with charged or polar residues. Our modeling revealed that Pro curves the peptide through a hinge, facilitating its insertion into the bacterial membrane and, once inserted, contributes to stabilizing the pore structure. The phylogenetic inference using hylid prepro-peptides showed the need to classify new AMPs using the full-length sequence of the prepro-peptide region and highlighted the complex relationships between peptide families. Our findings revealed that conserved motifs occurred independently in distinct AMP families, suggesting a convergent evolution and a significant role in peptide-membrane interactions.

Environmental and biological significance of the action of viper poison on the homeostasis of mammals

The article examines the ecological and biological significance of the action of animal poisons as an evolutionary adaptation that maximizes the adaptation of more than 250,000 species to the conditions of their habitat. It was found that animal poisons have a significant impact on the formation and course of various forms of connections in ecosystems, perform several ecological functions and are important biotic factors of the environment. The best-studied venomous animals have been found to be snakes, of over 3,800 different species, only a small number are highly venomous. The Viperidae family includes 101 species of poisonous snakes. In Europe, Vipera ammodytes, Vipera aspis, Vipera berus, Vipera latastei, Vipera seoanei and Vipera ursinii are the most dangerous species, their bites cause severe poisoning. In Ukraine, the genus Vipera is represented by the steppe viper (Vipera renardi (Cristoph, 1861)) and two subspecies of the common viper (Vipera berus (Linnaeus, 1758)) — Vipera berus berus and Nikolsky’s viper (Vipera berus nikolskii, Vedmederja Grubant et Rudaeva, 1986). The factors that affect the diversity of the composition of snake venom are given. It is noted that snake venoms are complex mixtures of proteins, peptides and low molecular weight substances, among which enzymes and non-enzymatic components are distinguished. The biological properties of snake venom depend significantly on the properties of its components, which have a wide range of effects on the general state of homeostasis of a living organism. A generalization of scientific data on the study of the chemical composition of the venom of Vipera renardi, Vipera berus nikolskii and Vipera berus berus and the effects of these venoms on the functioning of the mammalian body has been carried out. It was found that the venom of Vipera berus nikolskii is the most, and Vipera renardi — the least toxic for mammals (mice). The effect of Vipera berus nikolskii and Vipera berus berus venom on the proteolytic balance of individual organs of rats was evaluated. It is emphasized that viper venom toxins have a great, not yet fully developed potential in many scientific fields, therefore they deserve further practical research.

In Vitro Investigation of the Antibacterial Activity of Salamander Skin Peptides.

Given the current and future costs of antibiotic-resistant bacteria to human health and economic productivity, there is an urgent need to develop new antimicrobial compounds. Antimicrobial peptides are a promising alternative to conventional antibiotics and other antimicrobials. Amphibian skin is a rich source of bioactive compounds, but the antibacterial properties of salamander skin peptides have been neglected. Here, we examined the in vitro ability of skin peptides from 9 species of salamander representing 6 salamander families to inhibit the growth of ESKAPE pathogens, which are bacteria that have developed resistance to conventional antibiotics. We also examined whether the skin peptides caused lysis of human red blood cells. Skin peptides from Amphiuma tridactylum had the greatest antimicrobial properties, completely inhibiting the growth of all bacterial strains except for Enterococcus faecium. Likewise, skin peptides from Cryptobranchus alleganiensis completely inhibited the growth of several of the bacterial strains. In contrast, skin peptide mixtures from Ambystoma maculatum, Desmognathus fuscus, Eurycea bislineata, E. longicauda, Necturus beyeri, N. maculosus, and Siren intermedia did not completely inhibit bacterial growth even at the highest concentrations. Finally, none of the skin peptide mixtures caused lysis of human red blood cells. Together, we demonstrate that salamander skin produces peptides with potent antibacterial properties. It remains to elucidate the peptide sequences and their antibacterial mechanisms.

Antioxidant Effect Assessment and Trans Epithelial Analysis of New Hempseed Protein Hydrolysates.

Hempseed (Cannabis sativa) is one of the most promising sources of plant proteins. It contains approximately 24% (w/w) protein, and edestin accounts for approximately 60-80% (w/w) of its total proteins. In a framework of research aimed at fostering the proteins recovered from the press cake by-products generated after the extraction of hempseed oil, two hempseed protein hydrolysates (HH1 and HH2) were produced at an industrial level using a mixture of different enzymes from Aspergillus niger, Aspergillus oryzae, and Bacillus licheniformis for different times (5 h and 18 h). Using a combination of different direct antioxidant tests (DPPH, TEAC, FRAP, and ORAC assays, respectively), it has been demonstrated that HHs exert potent, direct antioxidant activity. A crucial feature of bioactive peptides is their intestinal bioavailability; for this reason, in order to solve this peculiar issue, the ability of HH peptides to be transported by differentiated human intestinal Caco-2 cells has been evaluated. Notably, by using mass spectrometry analysis (HPLC Chip ESI-MS/MS), the stable peptides transported by intestinal cells have been identified, and dedicated experiments confirmed that the trans-epithelial transported HH peptide mixtures retain their antioxidant activity, suggesting that these hempseed hydrolysates may be considered sustainable antioxidant ingredients to be exploited for further application, i.e., nutraceutical and/or food industries.

Reversed HILIC Gradient: A Powerful Strategy for On-Line Comprehensive 2D-LC.

The aim of the present work is to evaluate the possibilities and limitations of reversed hydrophilic interaction chromatography (revHILIC) mode in liquid chromatography (LC). This chromatographic mode consists of combining a highly polar stationary phase (bare silica) with a gradient varying from very low (1-5%) to high (40%) acetonitrile content (reversed gradient compared to HILIC). The retention behavior of revHILIC was first compared with that of reversed-phase LC (RPLC) and HILIC using representative mixtures of peptides and pharmaceutical compounds. It appears that the achievable selectivity can be ranked in the order RPLC > revHILIC > HILIC with the two different samples. Next, two-dimensional liquid chromatography (2D-LC) conditions were evaluated by combining RPLC, revHILIC, or HILIC with RPLC in an on-line comprehensive (LC × LC) mode. evHILIC × RPLC not only showed impressive performance in terms of peak capacity and sensitivity, but also provided complementary selectivity compared to RPLC × RPLC and HILIC × RPLC. Indeed, both the elution order and the retention time range differ significantly between the three techniques. In conclusion, there is no doubt that revHILIC should be considered as a viable option for 2D-LC analysis of small molecules and also peptides.

Gigaohm and Teraohm Resistors in Femtoamp and Picoamp Electrospray Ionization

The femtoamp electrospray ionization (femtoESI) mode has been shown to exhibit unique characteristics that may facilitate ionization efficiency studies and experiments requiring low ion beam flux. Investigation of femtoESI was hindered by a tiny, applied voltage window of 10-100 V, beyond which ionization currents quickly jumped to nanoamps. This window was difficult to locate because the exact onset voltage fluctuates due to variations in ion source alignments. Large resistors (0.1-100 TΩ) in series effectively expanded the femtoESI applied voltage range, up to 1400 V. By swapping resistors, rapid alternation allows for the comparison of both ESI modes under the same alignment. In peptide mixtures, analytes with lower surface activity are suppressed in the nanoESI mode whereas the femtoESI mode shows signal enhancement of less surface-active species. For protein solutions, there is little change in the charge states generated but the femtoESI mode does show a decrease in the average charge state of protein peaks. Peptides and proteins analyzed in the femtoESI mode also tend to generate higher intensity sodiated peaks over protonated peaks at specific charge states compared with nanoESI mode operation.

Identification of cardiorespiratory toxic components of Nemopilema nomurai jellyfish venom using sequential chromatography methods

Jellyfish stings pose a significant threat to humans in coastal areas worldwide, with venomous jellyfish species stinging millions of individuals annually. Nemopilema nomurai is one of the largest jellyfish species, with numerous tentacles rich in nematocysts. N. nomurai venom (NnV) is a complex mixture of proteins, peptides, and small molecules that serve as both prey-capture and defense mechanisms. Yet, the molecular identity of its cardiorespiratory and neuronal toxic components of NnV has not been clearly identified yet. Here, we isolated a cardiotoxic fraction, NnTP (Nemopilema nomurai toxic peak), from NnV using chromatographic methods. In the zebrafish model, NnTP exhibited strong cardiorespiratory and moderate neurotoxic effects. LC-MS/MS analysis identified 23 toxin homologs, including toxic proteinases, ion channel toxins, and neurotoxins. The toxins demonstrated a synergistic effect on the zebrafish, leading to altered swimming behavior, hemorrhage in the cardiorespiratory region, and histopathological changes in organs such as the heart, gill, and brain. These findings provide valuable insights into the mechanisms underlying the cardiorespiratory and neurotoxic effects of NnV, which could be useful in developing therapeutic strategies for venomous jellyfish stings.

Elucidating the mechanisms underlying the action of kenaf seed peptides mixture against gram-positive and gram-negative bacteria and its efficacy in whole milk preservation

The present study determined the in-situ antibacterial effect of cationic kenaf seed peptides mixture (KSPM) in whole milk and further comprehensively elucidated the mechanisms underlying the bactericidal action against gram-positive and gram-negative bacteria. The application experiments in artificially contaminated milk showed that KSPM at 0.5 and 1 mg/mL displayed a stable and strong bactericidal activity within 1–6 h of treatment and lasted up to two weeks storage at room and refrigeration conditions. The in-vitro mechanistic study showed that the KSPM underwent conformational changes from random coil to α-helix and β-sheet in the membrane-mimetic environment. The KSPM exerted its inhibitory effect in a concentration and time-dependent manner through cell membrane permeabilisation associated with membrane-disrupting effects, which is characterised by the cellular material leakage. The in-silico study revealed that the peptides efficiently interacted with bacterial peptidoglycan via hydrogen bonding, electrostatic interactions and hydrophobic forces. The peptide RALPSEHNK interacted with gram-positive and gram-negative bacterial phospholipid-phosphate head group mainly through electrostatic interaction. The current work indicated that KSPM exerts its bactericidal effect by membrane-targeted mechanism and it would be a promising natural candidate to control bacterial contamination in milk and dairy-based products.

Straightforward Creation of Possibly Prebiotic Complex Mixtures of Thiol-Rich Peptides.

At the origin of life, extremely diverse mixtures of oligomers and polymers could be obtained from relatively simple molecular bricks. Here, we present an example of the polymerization of two amidonitriles derived from cysteine, Cys-Ala-CN and Cys-Met-CN. The thiol function in a molecule adds onto the nitrile group of another one, allowing efficient condensation reactions and making available an extensive range of polymers containing amide bonds and/or five-membered heterocycles, namely thiazolines. Macrocycles were also identified, the biggest one containing sixteen residues (cyclo(Cys-Met)8). MALDI-TOF mass spectrometry was used to identify all the present species. What these examples show is that complex mixtures are likely to have formed on the primitive Earth and that, ultimately, the selection that must have followed may have been an even more crucial step towards life than the synthesis of the pre-biological species themselves.

Study on Immunity-enhancing Function of Golden Globular Immune Protein Factor

Golden globular immune protein factor is a mixture of peptides that can enhance personal immunity. The golden globular immune protein factor is mainly composed of cod peptide, milk fat globular membrane, immunoglobulin, lactoferrin and bovine thymosin peptide raw materials. This article mainly reviews the structure and components of the golden globular immune protein factor, and the application of the main components in the middle-aged and elderly people.

An optimized workflow for MS-based quantitative proteomics of challenging clinical bronchoalveolar lavage fluid (BALF) samples

BackgroundClinical bronchoalveolar lavage fluid (BALF) samples are rich in biomolecules, including proteins, and useful for molecular studies of lung health and disease. However, mass spectrometry (MS)-based proteomic analysis of BALF is challenged by the dynamic range of protein abundance, and potential for interfering contaminants. A robust, MS-based proteomics compatible sample preparation workflow for BALF samples, including those of small and large volume, would be useful for many researchers.ResultsWe have developed a workflow that combines high abundance protein depletion, protein trapping, clean-up, and in-situ tryptic digestion, that is compatible with either qualitative or quantitative MS-based proteomic analysis. The workflow includes a value-added collection of endogenous peptides for peptidomic analysis of BALF samples, if desired, as well as amenability to offline semi-preparative or microscale fractionation of complex peptide mixtures prior to LC–MS/MS analysis, for increased depth of analysis. We demonstrate the effectiveness of this workflow on BALF samples collected from COPD patients, including for smaller sample volumes of 1–5 mL that are commonly available from the clinic. We also demonstrate the repeatability of the workflow as an indicator of its utility for quantitative proteomic studies.ConclusionsOverall, our described workflow consistently provided high quality proteins and tryptic peptides for MS analysis. It should enable researchers to apply MS-based proteomics to a wide-variety of studies focused on BALF clinical specimens.

Intact Transition Epitope Mapping—Serological Inspection by Epitope EXtraction (ITEM—SIX)

Precision medicine requests accurate serological inspections to precisely stratify patients for targeted treatment. Intact transition epitope mapping analysis proved surrogate seroconversion of a model organism's serum when spiked with a monoclonal murine anti-Ovalbumin antibody (mAb) with epitope resolution. Isolation of the IgG fraction from blood serum applied two consecutive protein precipitation steps followed by ultrafiltration and resulted in an ESI-MS analysis-ready IgG preparation. For epitope mapping by epitope extraction, the Ovalbumin antigen was digested with trypsin. After desalting, the peptide mixture was added to the ESI-MS-ready IgG preparation from mAb-spiked serum and the solution was incubated to form an immune complex between the Ovalbumin-derived epitope peptide and the anti-Ovalbumin mAb. Then, the entire mixture of proteins and peptides was directly electrosprayed. Sorting of ions in the mass spectrometer's gas phase, dissociation of the immune complex ions by collision-induced dissociation, and recording of the epitope peptide ion that had been released from the immune complex proved the presence of the anti-Ovalbumin mAb in serum. Mass determination of the complex-released epitope peptide ion with isotope resolution is highly accurate, guaranteeing high specificity of this novel analysis approach, which is termed Intact Transition Epitope Mapping-Serological Inspections by Epitope EXtraction (ITEM-SIX).

A Narrative Review of Acthar Gel for the Treatment of Myositis.

Idiopathic inflammatory myopathies (IIMs) are autoimmune disorders characterized by symmetric proximal muscle weakness and chronic inflammation, with an increased risk of morbidity and mortality. The current standard of care includes traditional immunosuppressive pharmacotherapies; however, some patients cannot tolerate or do not adequately respond to these therapies, highlighting the need for alternative treatments for refractory disease. Acthar® Gel (repository corticotropin injection) is a naturally sourced mixture of adrenocorticotropic hormone analogs and other pituitary peptides that has been approved by the US Food and Drug Administration since 1952 for use in patients with two subgroups of IIMs, dermatomyositis (DM) and polymyositis (PM). However, it has not been routinely used in the treatment of IIMs. While Acthar may induce steroidogenesis, it also has a steroid-independent mechanism of action by exerting immunomodulatory effects through the activation of melanocortin receptors on immune cells, such as macrophages, B cells, and T cells. Recent clinical trials, retrospective analyses, and case reports add to the growing evidence suggesting that Acthar may be effective in patients with DM and PM. Here we review the current evidence supporting the safety and efficacy of Acthar for the treatment of refractory DM and PM.

Optimisation and identification of anchovy antimicrobial peptides by enzymolysis and untargeted proteomic profiling

Natural antimicrobial peptides are promising antibiotics without inducing high bacteria resistance and have wide applications in the medical health and food industry. Herein, we prepared and optimised anchovy antimicrobial peptides (AAMPs) by degreasing and enzymolysis of anchovy extracts under different conditions. The enzymolysis mixtures at optimised preparation conditions exhibited concentration-dependent inhibitory activity against Staphylococcus aureus. Then, we applied the mass spectrometry-based proteomics analysis to globally profile the peptide constitutes within the active peptide mixture and finally, 4 unique peptides coming from 68 proteins were confidently identified. We compared the peptide sequences with the antimicrobial peptides (AMPs) database and synthesised 4 potential AAMPs. As a result, peptide GTEDELDKY exhibited inhibitory activity against Escherichia coli, and peptides DREGESGPS, YIGRPIPQ and YEDEINKR exhibited inhibitory activity against SA. Our work established a useful workflow for discovering novel AMPs from fish proteins, which may play important role in the development of effective antibiotics.

Ultra-Accurate Correlation between Precursor and Fragment Ions in Two-Dimensional Mass Spectrometry: Acetylated vs Trimethylated Histone Peptides

Two-dimensional mass spectrometry (2D MS) is a method for tandem mass spectrometry in which precursor and fragment ions are correlated by manipulating ion radii rather than by ion isolation. A 2D mass spectrum contains the fragmentation patterns of all analytes in a sample, acquired in parallel. We report ultrahigh-resolution narrowband 2D mass spectra of a mixture of two histone peptides with the same sequence, one of which carries an acetylation and the other a trimethylation (m/z 0.006 difference). We reduced the distance between data points in the precursor ion dimension and compared the accuracy of the precursor-fragment correlation with the resolving power. We manage to perform label-free quantification on the histone peptide mixture and show that precursor and fragment ions can be accurately correlated even though the precursor ions are not resolved. Finally, we show that increasing the resolution of a 2D mass spectrum in the precursor ion dimension too far can lead to a decline in the signal-to-noise ratio.

Lipidomic profiling of the Brazilian yellow scorpion venom: new insights into inflammatory responses following Tityus serrulatus envenomation

ABSTRACT Due to the high prevalence and clinical relevance, scorpionism is a critical public health issue in several Brazilian regions. Tityus serrulatus, commonly known as the Brazilian yellow scorpion, is the most venomous genus found in Brazilian fauna and associated with severe clinical manifestations such as localized pain, hypertension, sweating, tachycardia and complex hyperinflammatory responses. In general, T. serrulatus venom contains a complex mixture of active compounds, including proteins, peptides, and amino acids. Although knowledge of the protein fractions of scorpion venom is available, venom lipid components are not yet comprehensively known. The aim of the present study was to determine and characterize the lipid constituents/profile of the T. serratus venom utilizing liquid chromatography coupled with high-resolution mass spectrometry. Lipid species (164 in total) belonging to 3 different lipid categories, glycerophospholipids, sphingolipids, and glycerolipids, were identified. A further search on MetaCore/MetaDrug platform, which is based upon a manually curated database of molecular interactions, molecular pathways, gene-disease associations, chemical metabolism, and toxicity information, exhibited several metabolic pathways for 24 of previously identified lipid species, including activation of nuclear factor kappa B and oxidative stress pathways. Further several bioactive compounds, such as plasmalogens, lyso-platelet-activating factors, and sphingomyelins, associated with systemic responses triggered by T. serrulatus envenomation were detected. Finally, lipidomic data presented provide advanced and valuable information to better comprehend the mechanisms underlying the complex pathophysiology induced by T. serrulatus envenomation.

Contribution of proteins to ceramic membrane fouling at the early stage of membrane filtration

Membrane-based filtration is widely applied for water and wastewater treatment, while membrane fouling caused by contaminants such as proteins is an inevitable problem. Protein-containing mixtures or intact model proteins (e.g., bovine serum albumin, BSA) have been conventionally adopted and recognized as important membrane foulants. However, due to the complex composition of the protein-containing mixture or the inappropriate concentrations of model substances adopted, the real contribution of protein-like fractions toward ceramic membrane fouling remains unknown yet. In this work, the fouling behaviors of BSA and tryptones, representing the intact protein and the mixture of peptides, respectively, in a ceramic membrane microfiltration system were investigated. Both of them exhibited a negligible flux decrease and membrane fouling. For comparison, the fouling behaviors of the environmental protein-like substances (EP) extracted from different sludge sources were also examined. In addition, the experimental and simulation results demonstrate that all the EP samples were easier to aggregate and be adsorbed on the membrane surface than the two model proteins because the environmental protein-like substances had more unsaturated groups. However, those properties were insufficient to cause rapid membrane pore blockage or biocake formation compared with the previous studies. Both the model and actual protein fractions led to light membrane fouling, implying that proteins were not mainly responsible for membrane fouling at the early stage of ceramic membrane microfiltration.

Development of nanomaterial enabling highly sensitive surface-assisted laser desorption/ionization mass spectrometry peptide analysis.

Surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) is an approach derived from matrix-assisted laser desorption/ionization (MALDI)-MS which overcomes the drawbacks associated with the use of organic matrices required to co-crystallize with the analytes. Indeed, nanomaterials commonly used in SALDI-MS as inert surfaces to promote desorption/ionization (D/I) ensure straightforward direct deposition of samples while providing mass spectra with ions only related to the compound of interest. The objective of this study was to develop a novel SALDI-MS approach based on steel plates that are surfaces very rapidly and easily tuned to perform the most efficient peptide detection as possible. To compare the SALDI efficacy of such metal substrates, D/I efficiency and deposit homogeneity were evaluated according to steel plate fabrication processes. The studied surfaces were nanostructured steel plates that were chemically modified by perfluorosilane and textured according to different frequencies and laser writing powers. The capacity of each tested 100 surfaces was demonstrated by comparative analyses of a mixture of standard peptides (m/z 600-3000) performed with a MALDI-TOF instrument enabling MALDI, SALDI and imaging experiments. A peptide mix was used to screen the different surfaces depending on their D/I efficiency and their ability to ensure homogeneous deposit of the samples. For that purpose, deposition homogeneity was visualized owing to reconstructed ionic images from all protonated or sodiated ions of the 10 peptides constituting the standard mix. Seven surfaces were then selected satisfying the required D/I efficiency and deposit homogeneity criteria. Results obtained with these optimal surfaces were then compared with those recorded by MALDI-MS analyses used as references.

A strategy for assessing peak purity of pharmaceutical peptides in reversed-phase chromatography methods using two-dimensional liquid chromatography coupled to mass spectrometry. Part I: Selection of columns and mobile phases

The current study describes the development of a 2D-LC-MS-based strategy for assessing main peak purity in the analysis of pharmaceutical peptides. The focus is on 2D-LC using reversed-phase (RP) separations in both dimensions, and particularly peptide isomer selectivity, since compounds with the same mass to charge ratio are not readily differentiated by mass spectrometry and therefore must be separated chromatographically. Initially, 30 column / mobile phase combinations were evaluated for both general separation performance (i.e., selectivity and peak shape) and isomer selectivity using forcibly degraded peptide samples and mixtures of synthetic diastereomers. A ranking of more than 300 UV and MS chromatograms suggests that when developing a new method, screening a set of four columns and four volatile mobile phases with differing characteristics should be adequate to both cover the selectivity space, and yield good separation performance. When 2D-LC-MS is to be used to evaluate peak purity for a new method, our results show that a second-dimension separation comprising a C8/C18 column possessing no ionic functionality, and an acetic acid / ammonium acetate mobile phase buffered at pH 5, provides good selectivity at 25 °C for peptide isomers with a MW <10 kDa.Retention data for 29 diverse peptides (1 < MW < 14 kDa, 3.7 < pI < 12.5) measured in this study using a variety of column and mobile phase conditions (i.e., 30 in total) are consistent with the classification of these various chromatographic conditions using the previously reported Peptide RPC Column Characterisation Protocol. For the investigated peptides trifluoroacetic acid was found to reduce selectivity differences between columns of diverse properties, probably due to its potential to form ion-pairs with peptides. Trifluoroacetic acid often improves peak shape for very large peptides (i.e. MW > 10 kDa). In the current dataset which also contain smaller peptides it received the highest ranking for 40% of the column and mobile phase combinations due to better selectivity and/or peak shape.The reported work here constitutes part one of a series of two papers. The second paper focuses on the use of retention modelling for rapid and accurate selection of the shallow gradients (i.e., << 1% ACN/min) required to obtain sufficient peptide isomer retention and separation in the second dimension. The overall results presented in this series of papers provides the guidance needed to develop a 2D-LC-MS method from start to finish for the analysis of main peak purity of therapeutic peptides.