Peptide Retention Research Articles

Peptide retention time prediction for electrostatic repulsion-hydrophilic interaction chromatography

Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) is one of the legacy separation tools developed by Dr. Andrew Alpert and has been used for developing unique separation methods of hydrophilic compounds, including peptides. In the past it has been studied using designed peptide libraries to elucidate major features of its separation mechanism, while comprehensive peptide retention modeling for ERLIC is still lacking. In this work we employed a proteomics-derived ∼170,000 peptide retention datasets to evaluate major ERLIC retention features using the framework of our Sequence-Specific Retention Calculator model. The separation conditions were adjusted to obtain a wider proteome coverage, particularly for non-modified peptides, resulting in a superior separation orthogonality for a 2D LC combination with reversed-phase C18 LC-MS in the second dimension. The SSRCalc ERLIC model presents a consistent theme with the existing ERLIC retention mechanism, reflecting a dependence on peptide orientation and the position of charged and hydrophilic residues across the peptide backbone. R2 values of 0.935 and 0.955 accuracy were demonstrated for the standard interpretable SSRCalc model and machine learning algorithm, respectively. The effects of various PTMs on peptide retention were evaluated in this study, covering spontaneous (oxidation, deamidation) and enzymatic (N-terminal acetylation, phosphorylation, glycosylation) modifications.

Peptide retention time prediction for hydrophilic interaction liquid chromatography at acidic pH in formic-acid based eluents

Peptide separation selectivity was evaluated for hydrophilic interaction liquid chromatography (HILIC) ZIC-HILIC, ZIC-cHILIC, and XBridge Amide sorbents using formic acid as eluent additive (pH 2.7). Sequence-specific retention prediction algorithms were trained using retention datasets of ∼30,000 peptides for each column. Our retention models were able to attain ∼0.98 R2-value and yielded retention coefficients that can be probed to understand peptide-stationary phase interaction. Overall, the hydrophilicity for these columns decreased when the mobile phase changed pH from 4.5 to 2.7, when using 0.1 % formic acid in the mobile phase. The acidic residues became protonated, and the resultant hydrophilic interaction is dampened at the lower pH, leaving only the basic residues as the primary hydrophilic interactors. Hence, peptides of increasing charge have higher retention. In this comparison between the three columns, ZIC-HILIC has the highest chromatographic resolution between groups of peptides of different charge. From the position-dependent retention coefficients for ZIC-HILIC at pH 2.7, we found that the amino acids at the terminal positions of the peptide modulate the basicity of the N-terminal amino group or the C-terminal Arg/Lys for tryptic peptides. With respect to the separation orthogonality between HILIC and acidic pH RPLC for two dimensional separations, the orthogonality values were lower at pH 2.7 than operating HILIC at pH 4.5 for the first dimension. We also demonstrate that ZIC-HILIC was able to distinguish citrullinated and deamidated peptides based on predicted retention values.

Calib-RT: an open source python package for peptide retention time calibration in DIA mass spectrometry data.

The data independent acquisition (DIA) mass spectrometry (MS) method is increasingly popular in the field of proteomics. But the loss of the correspondence between peptide ions and their spectra in DIA makes the identification challenging. One effective approach to reduce false positive identification is to calculate the deviation between the peptide's estimated retention time (RT) and measured RT. During this process, scaling the spectral library RT into the estimated RT, known as the RT calibration, is a prerequisite for calculating the deviation. Currently, within the DIA algorithm ecosystem, there is a lack of engine-independent and readily usable RT calibration toolkits. In this work, we introduce Calib-RT, a RT calibration method tailored to the characteristics of RT data. This method can achieve the nonlinear calibration across various data scales and tolerate a certain level of noise interference. Calib-RT is expected to enrich the open source DIA algorithm toolchain and assist in the development of DIA identification algorithms. Calib-RT is released as an open source software under the MIT license and can be installed from PyPi as a python module. The source code is available on GitHub at https://github.com/chenghui03/Calib_RT.

Paradigm Shift: Major Role of Ion-Pairing-Dependent Size Exclusion Effects in Bottom-Up Proteomics Reversed-Phase Peptide Separations.

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

Negative-Ion Electron Capture Dissociation of MALDI-Generated Peptide Anions.

Negative-ion electron capture dissociation (niECD) is an anion MS/MS technique that provides fragmentation analogous to conventional ECD, including high peptide sequence coverage and retention of labile post-translational modifications (PTMs). niECD has been proposed to be the most efficient for salt-bridged zwitterionic precursor ion structures. Several important PTMs, e.g., sulfation and phosphorylation, are acidic and can, therefore, be challenging to characterize in the positive-ion mode. Furthermore, PTM-friendly techniques, such as ECD, require multiple precursor ion-positive charges. By contrast, singly charged ions, refractory to ECD, are most compatible with niECD. Because electrospray ionization (ESI) typically yields multiply charged ions, we sought to explore matrix-assisted laser desorption/ionization (MALDI) in combination with niECD. However, the requirement for zwitterionic gaseous structures may preclude efficient niECD of MALDI-generated anions. Unexpectedly, we found that niECD of anions from MALDI is not only possible but proceeds with similar or higher efficiency compared with ESI-generated anions. Matrix selection did not appear to have a major effect. With MALDI, niECD is demonstrated up to m/z ∼4300. For such larger analytes, multiple electron captures are observed, resulting in triply charged fragments from singly charged precursor ions. Such charge-increased fragments show improved detectability. Furthermore, significantly improved (∼20-fold signal-to-noise increase) niECD spectral quality is achieved with equivalent sample amounts from MALDI vs ESI. Overall, the reported combination with MALDI significantly boosts the analytical utility of niECD.

A Mass Spectrometry Strategy for Protein Quantification Based on the Differential Alkylation of Cysteines Using Iodoacetamide and Acrylamide.

Mass spectrometry has become the most prominent yet evolving technology in quantitative proteomics. Today, a number of label-free and label-based approaches are available for the relative and absolute quantification of proteins and peptides. However, the label-based methods rely solely on the employment of stable isotopes, which are expensive and often limited in availability. Here we propose a label-based quantification strategy, where the mass difference is identified by the differential alkylation of cysteines using iodoacetamide and acrylamide. The alkylation reactions were performed under identical experimental conditions; therefore, the method can be easily integrated into standard proteomic workflows. Using high-resolution mass spectrometry, the feasibility of this approach was assessed with a set of tryptic peptides of human serum albumin. Several critical questions, such as the efficiency of labeling and the effect of the differential alkylation on the peptide retention and fragmentation, were addressed. The concentration of the quality control samples calculated against the calibration curves were within the ±20% acceptance range. It was also demonstrated that heavy labeled peptides exhibit a similar extraction recovery and matrix effect to light ones. Consequently, the approach presented here may be a viable and cost-effective alternative of stable isotope labeling strategies for the quantification of cysteine-containing proteins.

Intensity and retention time prediction improves the rescoring of protein-nucleic acid cross-links.

In protein-RNA cross-linking mass spectrometry, UV or chemical cross-linking introduces stable bonds between amino acids and nucleic acids in protein-RNA complexes that are then analyzed and detected in mass spectra. This analytical tool delivers valuable information about RNA-protein interactions and RNA docking sites in proteins, both in vitro and in vivo. The identification of cross-linked peptides with oligonucleotides of different length leads to a combinatorial increase in search space. We demonstrate that the peptide retention time prediction tasks can be transferred to the task of cross-linked peptide retention time prediction using a simple amino acid composition encoding, yielding improved identification rates when the prediction error is included in rescoring. For the more challenging task of including fragment intensity prediction of cross-linked peptides in the rescoring, we obtain, on average, a similar improvement. Further improvement in the encoding and fine-tuning of retention time and intensity prediction models might lead to further gains, and merit further research.

Toward an Ultimate Solution for Peptide Retention Time Prediction: The Effect of Column Temperature on Separation Selectivity.

We studied the effect of the column temperature on the selectivity of reversed-phase peptide separation in bottom-up proteomics. The number of peptide identifications from 2 h liquid chromatography with tandem mass spectrometry (LC-MS/MS) acquisitions reaches a plateau at 45-55 °C, driven simultaneously by improved separation efficiency, a gradual decrease in peptide retention, and possible on-column degradation of peptides at elevated temperatures. Performing 2D LC-MS/MS acquisitions at 25, 35, 45, and 55 °C resulted in the identification of ∼100,000 and ∼120,000 unique peptides for nonmodified and tandem mass tags (TMT)-labeled samples, respectively. These peptide collections were used to investigate the temperature-driven retention features. The latter is governed by the specific temperature response of individual residues, peptide hydrophobicity and length, and amphipathic helicity. On average, peptide retention decreased by 0.56 and 0.5% acetonitrile for each 10 °C increase for label-free and TMT-labeled peptides, respectively. This generally linear response of retention shifts allowed the extrapolation of predictive models beyond the studied temperature range. Thus, (trap) column cooling from room temperature to 0 °C will allow the retention of an additional 3% of detectable tryptic peptides. Meanwhile, the application of 90 °C would result in the loss of ∼20% of tryptic peptides that were amenable to MS/MS-based identification.

Impact of different spacers on the conjugation between Anderson-Evans polyoxometalates and peptides

The Anderson-Evans polyoxometalates (POM) display a promising anticancer activity. The conjugation with the GRP-receptor antagonist peptide Demobesin (fQWAVGHL-NHEt) was exploited to impart cell targeting capabilities and improve the selectivity of such polyanions. However, the POM interacts with the grafted peptides, inducing chains folding and self-assembly of the resulting hybrids, thus decreasing their recognition ability. Within this context, a tailored spacer, including two domains, i.e., a hydrophilic one (1,13-diamino-4,7,10-trioxatridecan-succinamic acid, Ttds) and a tetra-anionic one (Glu-Glu-Glu-Glu-βAla, EEEE-βA) was previously utilized to mitigate such interaction. In this work, hybrid POMs containing only Ttds or EEEE-βA were prepared and the contribution of the two spacers was separately studied by using 2D NMR, fluorimetry and circular dichroism (CD). Transmission electron microscopy (TEM) was also used to observe the impact of the different spacers on self-assembly. Owing to the relevant effects observed for EEEE-βA, MD calculations were finally performed to elucidate its behavior when incorporated in the hybrid POM. Our results show that, despite the stronger impact of EEEE-βA spacer, only when both spacer are present together it is possible to observe a significant effect on the retention of peptide's secondary structure and recognition capability.

Biomimetic multilayer scaffolds with prolonged retention of stem cells-recruiting and angiogenic peptides for promoting bladder regeneration

The complications of enterocystoplasy have challenged traditional bladder reconstruction methods. Tissue engineering scaffolds, involving stem cells implantation and vascularization strategies, emerge as promising alternatives. However, their applications are hindered by issues regarding the source of stem cells and the rapid loss of bioactive factors in a urinary environment. Here, a multilayer biomimetic scaffold consisting of polycaprolactone (PCL) nanofibrous mats and oxidized dextran/carboxymethyl chitosan hydrogel has been synthesized. Bone marrow homing peptide (BMHP) and vascular endothelial growth factor-mimicking peptide (VP) were incorporated into the hydrogel to recruit endogenous stem cells and promote vascularization, respectively. The results indicated that PCL mats not only reinforced the mechanical properties of composite scaffolds but also prolonged the retention of peptides via the hydrophobic surface. In vivo experiments demonstrated that BMHP and VP had crosstalk effects, amplifying their functions to accelerate bladder regeneration, enhance contractility of smooth muscle cells, inhibit fibrosis, and improve innervation. Overall, the novel functionalized scaffolds are potentially viable for urologic tissue reconstruction.

Polylactic Glycolic Acid-Mediated Delivery of Plectasin Derivative NZ2114 in Staphylococcus epidermidis Biofilms.

Antimicrobial peptides (AMPs) are antibiotic candidates; however, their instability and protease susceptibility limit clinical applications. In this study, the polylactic acid-glycolic acid (PLGA)-polyvinyl alcohol (PVA) drug delivery system was screened by orthogonal design using the double emulsion-solvent evaporation method. NZ2114 nanoparticles (NZ2114-NPs) displayed favorable physicochemical properties with a particle size of 178.11 ± 5.23 nm, polydispersity index (PDI) of 0.108 ± 0.10, ζ potential of 4.78 ± 0.67 mV, actual drug-loading rate of 4.07 ± 0.37%, encapsulation rate of 81.46 ± 7.42% and cumulative release rate of 67.75% (120 h) in PBS. The results showed that PLGA encapsulation increased HaCaT cell viability by 20%, peptide retention in 50% serum by 24.12%, and trypsin tolerance by 4.24-fold. Meanwhile, in vitro antimicrobial assays showed that NZ2114-NPs had high inhibitory activity against Staphylococcus epidermidis (S. epidermidis) (4-8 μg/mL). Colony counting and confocal laser scanning microscopy (CLSM) confirmed that NZ2114-NPs were effective in reducing the biofilm thickness and bacterial population of S. epidermidis G4 with a 99% bactericidal rate of persister bacteria, which was significantly better than that of free NZ2114. In conclusion, the results demonstrated that PLGA nanoparticles can be used as a reliable NZ2114 delivery system for the treatment of biofilm infections caused by S. epidermidis.

Aptamer-Functionalized Interface Nanopores Enable Amino Acid-Specific Peptide Detection.

Single-molecule proteomics based on nanopore technology has made significant advances in recent years. However, to achieve nanopore sensing with single amino acid resolution, several bottlenecks must be tackled: controlling nanopore sizes with nanoscale precision and slowing molecular translocation events. Herein, we address these challenges by integrating amino acid-specific DNA aptamers into interface nanopores with dynamically tunable pore sizes. A phenylalanine aptamer was used as a proof-of-concept: aptamer recognition of phenylalanine moieties led to the retention of specific peptides, slowing translocation speeds. Importantly, while phenylalanine aptamers were isolated against the free amino acid, the aptamers were determined to recognize the combination of the benzyl or phenyl and the carbonyl group in the peptide backbone, enabling binding to specific phenylalanine-containing peptides. We decoupled specific binding between aptamers and phenylalanine-containing peptides from nonspecific interactions (e.g., electrostatics and hydrophobic interactions) using optical waveguide lightmode spectroscopy. Aptamer-modified interface nanopores differentiated peptides containing phenylalanine vs. control peptides with structurally similar amino acids (i.e., tyrosine and tryptophan). When the duration of aptamer-target interactions inside the nanopore were prolonged by lowering the applied voltage, discrete ionic current levels with repetitive motifs were observed. Such reoccurring signatures in the measured signal suggest that the proposed method has the possibility to resolve amino acid-specific aptamer recognition, a step toward single-molecule proteomics.

Retention time prediction for post-translationally modified peptides: Ser, Thr, Tyr-phosphorylation

The development of a peptide retention prediction model for reversed-phase chromatography applications in proteomics is reported for peptides carrying phosphorylated Ser, Thr and Tyr-residues. The major retention features have been assessed using a collection of over 10,000 phosphorylated/non-phosphorylated peptide pairs identified in a series 1D and 2D LC-MS/MS acquisitions using formic acid as ion pairing modifier. Single modification event on average results in increased peptide retention for phosphorylation of Ser (+ 1.46), Thr (+1.33), Tyr (+0.93% acetonitrile, ACN) on gradient elution scale for Luna C18(2) stationary phase. We established several composition and sequence specific features, which drive deviations from these average values. Thus, single phosphorylation of serine results in retention shifts ranging from -2.4 to 5.5% ACN depending on position of the residue, nature of nearest neighbour residues, peptide length, hydrophobicity and pI value, and its propensity to form amphipathic helical structures. We established that the altered ion-pairing environment upon phosphorylation is detrimental for this variability. Hydrophobicity of ion-pairing modifier directly informs the magnitude of expected shifts: (most hydrophilic) 0.5 % acetic acid (larger positive shift upon phosphorylation) > 0.1 % formic acid (positive) > 0.1 % trifluoroacetic (negative) > 0.1 % heptafluorobutyric acid (larger negative shift). The effect of phosphorylation has been also evaluated for several separation conditions used in the first dimension of 2D LC applications: high pH reversed-phase (RP), hydrophilic interaction liquid chromatography (HILIC), strong cation- and strong anion exchange separations.

MARS an improved de novo peptide candidate selection method for non-canonical antigen target discovery in cancer

Understanding the nature and extent of non-canonical human leukocyte antigen (HLA) presentation in tumour cells is a priority for target antigen discovery for the development of next generation immunotherapies in cancer. We here employ a de novo mass spectrometric sequencing approach with a refined, MHC-centric analysis strategy to detect non-canonical MHC-associated peptides specific to cancer without any prior knowledge of the target sequence from genomic or RNA sequencing data. Our strategy integrates MHC binding rank, Average local confidence scores, and peptide Retention time prediction for improved de novo candidate Selection; culminating in the machine learning model MARS. We benchmark our model on a large synthetic peptide library dataset and reanalysis of a published dataset of high-quality non-canonical MHC-associated peptide identifications in human cancer. We achieve almost 2-fold improvement for high quality spectral assignments in comparison to de novo sequencing alone with an estimated accuracy of above 85.7% when integrated with a stepwise peptide sequence mapping strategy. Finally, we utilize MARS to detect and validate lncRNA-derived peptides in human cervical tumour resections, demonstrating its suitability to discover novel, immunogenic, non-canonical peptide sequences in primary tumour tissue.

Cancer Cell Targeting Via Selective Transferrin Receptor Labeling Using Protein-Derived Carbon Dots.

Carbon dot (CD) nanoparticles offer tremendous advantages as fluorescent probes in bioimaging and biosensing; however, they lack specific affinity for biomolecules, limiting their practical applications in selective targeting. Nanoparticles with intrinsic affinity for a target have applications in imaging, cytometry, therapeutics, etc. Toward that end, we report the transferrin receptor (CD71) targeting CDs, synthesized for the first time. The formation of these particles is truly groundbreaking, as direct tuning of nanoparticle affinity was achieved by simple and careful precursor selection of a protein, which has the targeting characteristic of interest. We hypothesized that the retention of the original protein's peptides on the nanoparticle surface provides the CDs with some of the function of the precursor protein, enabling selective binding to the protein's receptor. This was confirmed with FTIR (Fourier transform infrared) data and subsequent affinity-based cell assays. These transferrin (Tf)-derived CDs have been shown to possess an affinity for CD71, a cancer biomarker that is ubiquitously expressed in nearly every cancer cell line due to its central role mediating the uptake of cellular iron. The CDs were tested using the human leukemia cell line HL60 and demonstrated the selective targeting of CD71 and specific triggering of transferrin-mediated endocytosis via clathrin-coated pits. The particle characterization results reflect a carbon-based nanoparticle with bright violet fluorescence and 7.9% quantum yield in aqueous solution. These unpresented CDs proved to retain the functional properties of the precursor protein. Indicating that this process can be repeated for other disease biomarkers for applications ranging from biosensing and diagnostic bioimaging to targeted therapeutics.

Fatty acids-modified liposomes for encapsulation of bioactive peptides: Fabrication, characterization, storage stability and in vitro release

The fragile membranes of liposomes limit their application by the food industry. In this study, we hypothesized that interactions between fatty acids with different chain lengths and phospholipids might enhance liposome stability. Decanoic acid modified liposomes (Lipo-DA) and stearic acid modified liposomes (Lipo-SA) were fabricated for encapsulation of hydrophilic peptides. Fluorescence spectroscopy and FTIR analysis showed molecular interactions existed between alkyl chains and phospholipids, resulting in greater compactness and hydrophobicity of the membranes in Lipo-DA and Lipo-SA. This led to a reduction in melting point characterized by differential scanning calorimetry analysis. Lipo-DA and Lipo-SA could delay the release of hydrophilic peptides compared with unmodified liposomes in simulated digestion. Moreover, Lipo-DA showed better stability during storage, while Lipo-SA exhibited precipitation, resulting in the lowest peptide retention. Our study showed that decanoic acid is suitable to enhance the stability of liposomes, although this approach has yet to be tested in food products.

UF fractionation of fish protein hydrolysate

A commercial fish protein hydrolysate was fractionated using polymeric spiral wound ultrafiltration (UF) membranes. As protein hydrolysates are electrolytes, the effects of pH and ionic strength were investigated. Three different polyethersulfones (PES) membranes with different nominal molecular weight cut-offs (10, 5, and 3 kDa) and a polyamide-thin film composite (PA) membrane with a nominal molecular weight cut-off of 3 kDa were used. The membrane flux and retention profile, and the selectivity for <4 kDa peptides were analyzed and discussed. The PES membranes were suitable for fractionation of the hydrolysate, while the PA membrane showed high retention for all components because of the adsorption of solutes to the membrane surface. The results showed that with charged solutes, the nominal molecular weight cut-off as supplied by manufacturers was not the decisive parameter for choosing a membrane. The permeate flux at pH 8 was higher than that at pH 5 for all PES membranes. The presence of salt in the hydrolysate significantly improved the flux and selectivity. The overall retention mainly was dependent on the retention of small peptides (<4 kDa). The mass transfer mechanism of charged peptides within the concentration polarization during UF separation was simplified and presented regarding size and charge exclusions.

Compendium of Chromatographic Behavior of Post-translationally and Chemically Modified Peptides in Bottom-Up Proteomic Experiments.

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

Characterization of LL37 Binding to Collagen through Peptide Modification with a Collagen-Binding Domain.

Collagen-based biomaterials loaded with antimicrobial peptides (AMPs) present a promising approach for promoting wound healing while providing protection against infections. In our previous work, we modified the AMP LL37 by incorporating a collagen-binding domain (cCBD) as an anchoring unit for collagen-based wound dressings. We demonstrated that cCBD-modified LL37 (cCBD-LL37) exhibited improved retention on collagen after washing with PBS. However, the binding mechanism of cCBD-LL37 to collagen remained to be elucidated. In this study, we found that cCBD-LL37 showed a slightly higher affinity for collagen compared to LL37. Our results indicated that cCBD inhibited cCBD-LL37 binding to collagen but did not fully eliminate the binding. This suggests that cCBD-LL37 binding to collagen may involve more than just one-site-specific binding through the collagen-binding domain, with non-specific interactions also playing a role. Electrostatic studies revealed that both LL37 and cCBD-LL37 interact with collagen via long-range electrostatic forces, initiating low-affinity binding that transitions to close-range or hydrophobic interactions. Circular dichroism analysis showed that cCBD-LL37 exhibited enhanced structural stability compared to LL37 under varying ionic strengths and pH conditions, implying potential improvements in antimicrobial activity. Moreover, we demonstrated that the release of LL37 and cCBD-LL37 into the surrounding medium was influenced by the electrostatic environment, but cCBD could enhance the retention of peptide on collagen scaffolds. Collectively, these results provide important insights into cCBD-modified AMP-binding mechanisms and suggest that the addition of cCBD may enhance peptide structural stability and retention under varying electrostatic conditions.

Assessing the hydrophobicity of glycopeptides using reversed-phase liquid chromatography and tandem mass spectrometry

Retention time is one of the most important parameters that has been widely used to demonstrate the separation results obtained from liquid chromatography (LC) platforms. However, retention time can shift when samples are tested with different instruments and laboratories, which hinders the identification process of analytes when comparing data collected from different LC systems. To address this problem, hydrophobicity index was introduced for retention time normalization of the glycopeptides separated by reversed-phase LC (RPLC). Tandem MS was used for the detection and identification of glycopeptides. In addition, the influence of different types of glycans on the hydrophobicity of peptide backbones was studied by comparing the retention time of glycopeptides with their non-glycosylated counterparts. The hydrophobicity of tryptic digested glycopeptides derived from model glycoproteins, including bovine fetuin, α1-acid glycoprotein, and haptoglobin from human plasma, were evaluated based on the hydrophobicity index of the standard peptides from a peptide retention time calibration mixture. The reduction of hydrophobicity of multiple peptide backbones was observed due to the hydrophilic glycan structures. By comparing the hydrophobicity index of glycopeptides collected from different time and instruments, the day-to-day and lab-to-lab comparisons suggested high reliability and reproducibility of this approach. The RSD% of hydrophobicity index from inter-lab experiments was 1.2%, while the RSD% of retention time was 5.1%. Then, the applications of this method were demonstrated on complex glycopeptide samples extracted from human blood serum. The hydrophobicity index can be applied to address the retention time shift when using different instruments, thereby boosting confidence of the characterization of glycopeptides.