Microfluidics, an effective tool for supporting phage display-A review.

Phage display is a vital tool for the discovery and development of affinity reagents such as antibodies and peptides, which have great potential in imaging, molecular recognition, biosensors, targeted delivery and other clinical applications. However, affinity reagents obtained by phage display are often subjected to a process called biopanning, which is considered time-consuming, labor-intensive and lacks accurate control, limiting the acquisition of high-quality affinity reagents. Over the last two decades, several microfluidic approaches have been designed to simplify the conventional biopanning process and to realize precise control. To better understand the advantages of microfluidics over traditional biopanning and the potential of microfluidics for other molecular screening strategies, we provided an overview of recent applications of microfluidics in phage display. Additionally, the next challenges and outlooks are discussed.

Qualitative and quantitative characterization of elastin-like polypeptides combining low-PH/low-temperature bottom-up and intact protein liquid chromatography mass spectrometric analysis.

RATIONALE: Elastin-like polypeptides (ELPs) are elastic and thermoresponsive biopolymers composed of VPGXG repeats (X can be any amino acid except proline), used in biomedical applications, for example, tissue engineering and drug delivery. As different variants of ELP are mostly produced fermentatively, there is a need for the development of analysis methods that allow for absolute protein quantification in both complex matrices and purified samples and MW determination of the final products. METHODS: ELPs were intracellularly expressed in Escherichia coli quantified after cell lysis and enzymatic digestion using a proline-specific protease ProAlanase (Promega) at acidic conditions. Resulting peptides were separated by liquid chromatography, and mass spectrometry analysis was conducted by electrospray ionization high-resolution mass spectrometry using an Orbitrap mass spectrometer. The addition of a stable isotopically labeled internal standard enabled quantification in complex matrices. Prior to intact mass analysis, ELPs were purified from fermentation broth by inverse temperature cycling. Intact protein analysis was performed using reversed-phase liquid chromatography, and mass spectrometry analysis was conducted by electrospray ionization high-resolution mass spectrometry using a time-of-flight mass spectrometer. RESULTS: Absolute quantification of ELPs was achieved by utilizing ELP-specific properties, that is, proline-rich, soluble at low pH and low temperature. The repetitive nature of ELPs allows for sensitivity increase and use of higher dilution factors to minimize the matrix effects. Despite the lack of amino acids with charged side chains (Arg, His, Lys, Asp, and Glu) in ELP, we demonstrated successful intact protein analysis using reversed-phase LC coupled to electrospray ionization TOF MS. Moreover, truncated protein forms could be chromatographically separated and characterized as well as N-terminal modifications. CONCLUSIONS: Both methods combined enabled quantitative and qualitative characterization of fermentatively produced ELPs.

Amino Acid Composition Determination From the Fractional Mass of Peptides.

A peptide's fractional mass is directly associated with its elemental composition and thus amino acid composition. Here it is demonstrated that a peptide's fractional mass alone can be a useful identifier or indicator of that composition for small to mid-sized peptides (5-7 amino acids) and can significantly reduce the number of viable amino acid compositions for larger peptides (> 8 residues) to include or exclude certain possibilities. Separate consideration of the integer portion of the peptide's mass helps to reduce the number of possibilities where many duplicate fractional mass values are found. Adoption of this fractional mass strategy should aid approaches that are presently employed for peptide identification, including in the use of mass map data to search protein databases for proteomics applications.

An optimised method to isotopically label pure synthetic peptides 'in-house' for absolute quantification in bottom-up proteomics.

RATIONALE: Heavy-labelled internal standards increasingly represent the gold standard for absolute quantitation in mass spectrometry (MS)-based bottom-up proteomics. The biggest drawbacks of using these standards are that they have high costs and lengthy lead times. METHODS: We describe an efficient, low-cost optimised method to enable 'in-house' heavy labelling of synthetic tryptic peptides for absolute quantification using tandem LC-MS/MS mass spectrometry. Our methodology uses 18O water in a trypsin-catalysed oxygen exchange reaction at the carboxyl terminus with the overall aim of reducing the costs and lead time associated with sourcing heavy standards from commercial vendors. RESULTS: Step-by-step instructions are provided on how to execute this protocol with high-throughput adaptations utilising a 96-well plate and a liquid-handling robot. Detailed notes on experimental setup, tips for troubleshooting and suggested improvements to maximise labelling efficiencies are highlighted to achieve the best results. Under optimum conditions, labelling efficiencies of peptides can reach from 95% to 100%. CONCLUSIONS: The application of the 'in-house' labelled standards in generating calibration curves to quantify endogenous peptide concentrations is just as effective as using the synthetically sourced standards while also having great cost reduction implications as well as saving time spent waiting for peptides to arrive. The protocol is highly adaptable and can be customized to fit the specific setup of any laboratory, maximizing achievable labelling efficiencies.

High-resolution quadrupole improves spectral purity and reduces interference from non-target ions in isobaric multiplexed quantitative proteomics.

BACKGROUND: Mass spectrometry (MS)-based proteomics is a powerful tool for identifying and quantifying proteins. However, chimeric spectra caused by the fragmentation of multiple precursors within the same isolation window impair the accuracy of peptide identification and isobaric mass tag-based quantification. While there have been advances in computational deconvolution of chimeric spectra and methods to further separate the peptides by ion mobility or through MSn, the use of narrower isolation windows to decrease the fraction of chimeric species remains to be fully explored. RESULTS: We present results obtained on a SCIEX TripleTOF instrument where the quadrupole was optimized and tuned for precursor isolation at 0.1 Da (FWHH). Using a three-proteome model (trypsin digest of protein lysates from yeast, human and E. coli) and 8-plex iTRAQ labeling to document the interference effect, we investigated the impact of co-fragmentation on spectral purity, identification accuracy and quantification accuracy. The narrow quadrupole isolation window significantly improved the spectral purity and reduced the interference of non-target precursors on quantification accuracy. The high-resolution isolation strategy also reduced the number of false identifications caused by chimeric spectra. While these improvements came at the cost of sensitivity loss, combining high-resolution isolation with other advanced techniques, including ion mobility, may result in improved accuracy in identification and quantification. SIGNIFICANCE: Compared to standard-resolution quadrupole isolation (0.7 Da), high-resolution quadrupole isolation (0.1 Da) significantly improved the spectral purity and quantification accuracy while reducing the number of potential false identifications caused by chimeric spectra, thus showing excellent potential for further development to analyze clinical proteomics samples, for which high accuracy is essential.

Coupling Microdroplet-Based Sample Preparation, Multiplexed Isobaric Labeling, and Nanoflow Peptide Fractionation for Deep Proteome Profiling of the Tissue Microenvironment.

There is increasing interest in developing in-depth proteomic approaches for mapping tissue heterogeneity in a cell-type-specific manner to better understand and predict the function of complex biological systems such as human organs. Existing spatially resolved proteomics technologies cannot provide deep proteome coverage due to limited sensitivity and poor sample recovery. Herein, we seamlessly combined laser capture microdissection with a low-volume sample processing technology that includes a microfluidic device named microPOTS (microdroplet processing in one pot for trace samples), multiplexed isobaric labeling, and a nanoflow peptide fractionation approach. The integrated workflow allowed us to maximize proteome coverage of laser-isolated tissue samples containing nanogram levels of proteins. We demonstrated that the deep spatial proteomics platform can quantify more than 5000 unique proteins from a small-sized human pancreatic tissue pixel (∼60,000 µm2) and differentiate unique protein abundance patterns in pancreas. Furthermore, the use of the microPOTS chip eliminated the requirement for advanced microfabrication capabilities and specialized nanoliter liquid handling equipment, making it more accessible to proteomic laboratories.

Analytical considerations for characterization of generic peptide product: A regulatory insight.

The Peptide therapeutics market was evaluated to be around USD 45.67 BN in 2023 and is projected to witness massive growth at a CAGR of around 5.63 % from 2024 to 2032 (USD 80.4 BN). Generic peptides are expected to reach USD 27.1 billion by 2032 after the patent monopoly of the pioneer peptides expires, and generic peptides become accessible. The generic manufacturers are venturing into peptide-based therapeutics for the aforementioned reasons. There is an abundance of material accessible regarding the characterization of peptides, which can be quite confusing for researchers. The FDA believes that an ANDA applicant may now demonstrate that the active component in a proposed generic synthetic peptide drug product is the "same" as the active ingredient in a peptide of rDNA origin that has previously been approved. To ensure the efficacy, safety, and quality of peptide therapies during development, regulatory bodies demand comprehensive characterization utilizing several orthogonal methodologies. This article elaborates the peptide characterization by segmenting into different segments as per the critical quality attribute from identification of the peptide to the physicochemical property of the peptide therapeutics which will be required to demonstrate the sameness with reference product based on the size of the peptide chain and molecular weight of the peptides. Article insights briefly on each individual technique and the orthogonal techniques for each test were explained. The impurities requirements in the generic peptides as per the regulatory requirement were also discussed.

CPred: Charge State Prediction for Modified and Unmodified Peptides in Electrospray Ionization.

The mass-to-charge ratio serves as a critical parameter in peptide identification via mass spectrometry, enabling the precise determination of peptide masses and facilitating their differentiation based on unique charge characteristics, especially when peptides are ionized by tools like electrospray ionization, which produces multiply charged ions. We developed a neural network called CPred, which can accurately predict the charge state distribution from +1 to +7 for the modified and unmodified peptides. CPred was trained on the large-scale synthetic training data, consisting of tryptic and non-tryptic peptides, and various fragmentation methods. The model was further evaluated on independent, external test data sets. Results were evaluated through the Pearson correlation coefficient and showed high correlations of up to 0.9997117 between the predicted and acquired charge state distributions. The effect of specifying modifications in the neural network and feature importance was further investigated, revealing the value of modifications and vital peptide properties in holding on to protons. CPreds' accurate predictions of the charge state distribution can play an essential role in boosting confidence in peptide identifications during rescoring as a novel feature.

Non-human peptides revealed in blood reflect the composition of intestinal microbiota.

BACKGROUND: The previously underestimated effects of commensal gut microbiota on the human body are increasingly being investigated using omics. The discovery of active molecules of interaction between the microbiota and the host may be an important step towards elucidating the mechanisms of symbiosis. RESULTS: Here, we show that in the bloodstream of healthy people, there are over 900 peptides that are fragments of proteins from microorganisms which naturally inhabit human biotopes, including the intestinal microbiota. Absolute quantitation by multiple reaction monitoring has confirmed the presence of bacterial peptides in the blood plasma and serum in the range of approximately 0.1 nM to 1 µM. The abundance of microbiota peptides reaches its maximum about 5 h after a meal. Most of the peptides correlate with the bacterial composition of the small intestine and are likely obtained by hydrolysis of membrane proteins with trypsin, chymotrypsin and pepsin - the main proteases of the gastrointestinal tract. The peptides have physicochemical properties that likely allow them to selectively pass the intestinal mucosal barrier and resist fibrinolysis. CONCLUSIONS: The proposed approach to the identification of microbiota peptides in the blood, after additional validation, may be useful for determining the microbiota composition of hard-to-reach intestinal areas and monitoring the permeability of the intestinal mucosal barrier.

Determination of amino acids and peptides without their pre-column derivatization by capillary electrophoresis with ultraviolet and contactless conductivity detection. An overview.

This review provides an overview of recent works focusing on the determination of amino acids (AAs) and peptides using capillary electrophoresis with contactless conductivity detection and ultraviolet (UV) detection, which is the most widespread detection in capillary electromigration techniques, without pre-capillary derivatization. Available options for the UV detection of these analytes, such as indirect detection, complexation with transition metal ions, and in-capillary derivatization are described. Developments in the field of direct detection of UV-absorbing AAs and peptides as well as progress in chiral separation are described. A separate section is dedicated to using on-line sample preconcentration methods combined with capillary electrophoresis-UV.

A New Evaluation Metric for Quantitative Accuracy of LC-MS/MS-Based Proteomics with Data-Independent Acquisition.

Data-independent acquisition (DIA) has improved the identification and quantitation coverage of peptides and proteins in liquid chromatography-tandem mass spectrometry-based proteomics. However, different DIA data-processing tools can produce very different identification and quantitation results for the same data set. Currently, benchmarking studies of DIA tools are predominantly focused on comparing the identification results, while the quantitative accuracy of DIA measurements is acknowledged to be important but insufficiently investigated, and the absence of suitable metrics for comparing quantitative accuracy is one of the reasons. A new metric is proposed for the evaluation of quantitative accuracy to avoid the influence of differences in false discovery rate control stringency. The part of the quantitation results with high reliability was acquired from each DIA tool first, and the quantitative accuracy was evaluated by comparing quantification error rates at the same number of accurate ratios. From the results of four benchmark data sets, the proposed metric was shown to be more sensitive to discriminating the quantitative performance of DIA tools. Moreover, the DIA tools with advantages in quantitative accuracy were consistently revealed by this metric. The proposed metric can also help researchers in optimizing algorithms of the same DIA tool and sample preprocessing methods to enhance quantitative accuracy.

Identification and screening of umami peptides from skipjack tuna (Katsuwonus pelamis) hydrolysates using EAD/CID based micro-UPLC-QTOF-MS and the molecular interaction with T1R1/T1R3 taste receptor.

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.

Exploring bioactive compounds in chickpea and bean aquafaba: Insights from glycomics and peptidomics analyses.

The objective of this study was to identify bioactive oligosaccharides and peptides in the cooking water of chickpeas and common beans, known as aquafaba. The oligosaccharides stachyose, raffinose and verbascose were quantified by high-performance anion-exchange chromatography; 78 and 67 additional oligosaccharides were identified in chickpea and common bean aquafaba, respectively, by LC-MS/MS. Chickpea aquafaba uniquely harbored ciceritol and other methyl-inositol-containing oligosaccharides. In prebiotic growth assays, chickpea aquafaba oligosaccharides were differentially utilized, promoting growth of Limosilactobacillus reuteri DSM 20016 and Bifidobacterium longum subsp. infantis ATCC 15697, but not Lacticaseibacillus rhamnosus GG. Dimethyl labeling, along with LC-MS/MS, effectively differentiated α- and γ-glutamyl peptides, revealing the presence of several γ-glutamyl peptides known to possess kokumi and anti-inflammatory activities, including γ-Glu-Phe and γ-Glu-Tyr in chickpeas aquafaba and γ-Glu-S-methyl-Cys and γ-Glu-Leu in beans aquafaba. This work uncovered unique bioactive peptides and oligosaccharides in aquafaba, helping promote its valorization, food system sustainability, and future health-promoting claims.

Mitigating In-Column Artificial Modifications in High-Temperature LC-MS for Bottom-Up Proteomics and Quality Control of Protein Biopharmaceuticals.

Elevating the column temperature is an effective strategy for improving the chromatographic separation of peptides. However, high temperatures induce artificial modifications that compromise the quality of the peptide analysis. Here, we present a novel high-temperature LC-MS method that retains the benefits of a high column temperature while significantly reducing peptide modification and degradation during reversed-phase liquid chromatography. Our approach leverages a short inline trap column maintained at a near-ambient temperature installed upstream of a separation column. The retentivity and dimensions of the trap column were optimized to shorten the residence time of peptides in the heated separation column without compromising the separation performance. This easy-to-implement approach increased peak capacity by 1.4-fold within a 110 min peptide mapping of trastuzumab and provided 10% more peptide identifications in exploratory LC-MS proteomic analyses compared with analyses conducted at 30 °C while maintaining the extent of modifications close to the background level. In the peptide mapping of biopharmaceuticals, where in-column modifications can falsely elevate the levels of some critical quality attributes, the method reduced temperature-related artifacts by 66% for N-terminal pyroGlu and 63% for oxidized Met compared to direct injection at 60 °C, thus improving reliability in quality control of protein drugs. Our findings represent a promising advancement in LC-MS methodology, providing researchers and industry professionals with a valuable tool for improving the chromatographic separation of peptides while significantly reducing the unwanted modifications.

Comparison of Peptidomes Extracted from Healthy Tissue and Tumor Tissue of the Parotid Glands and Saliva Samples.

Salivary gland tumors are highly variable in clinical presentation and histology. The World Health Organization (WHO) classifies 22 types of malignant and 11 types of benign tumors of the salivary glands. Diagnosis of salivary gland tumors is based on imaging (ultrasound, magnetic resonance imaging) and fine-needle aspiration biopsy, but the final diagnosis is based on histopathological examination of the removed tumor tissue. In this pilot study, we are testing a new approach to identifying peptide biomarkers in saliva that can be used to diagnose salivary gland tumors. The research material for the peptidomic studies was extracts from washings of neoplastic tissues and healthy tissues (control samples). At the same time, saliva samples from patients and healthy individuals were analyzed. The comparison of the peptidome composition of tissue extracts and saliva samples may allow the identification of potential peptide markers of salivary gland tumors in patients' saliva. The peptidome compositions extracted from 18 tumor and 18 healthy tissue samples, patients' saliva samples (11 samples), and healthy saliva samples (8 samples) were analyzed by LC-MS tandem mass spectrometry. A group of 109 peptides was identified that were present only in the tumor tissue extracts and in the patients' saliva samples. Some of the identified peptides were derived from proteins previously suggested as potential biomarkers of salivary gland tumors (ANXA1, BPIFA2, FGB, GAPDH, HSPB1, IGHG1, VIM) or tumors of other tissues or organs (SERPINA1, APOA2, CSTB, GSTP1, S100A8, S100A9, TPI1). Unfortunately, none of the identified peptides were present in all samples analyzed. This may be due to the high heterogeneity of this type of cancer. The surprising result was that extracts from tumor tissue did not contain peptides derived from salivary gland-specific proteins (STATH, SMR3B, HTN1, HTN3). These results could suggest that the developing tumor suppresses the production of proteins that are essential components of saliva.

Using peptide barcodes for simultaneous profiling of protein expression from mRNA.

RATIONALE: mRNA technology has begun to play a significant role in the areas of therapeutic intervention and vaccine development. However, optimizing the mRNA sequence that influences protein expression levels is a resource-intensive and time-consuming process. This study introduces a new method to accelerate the selection of sequences of mRNA for optimal protein expression. METHODS: We designed the mRNA sequences in such a way that a unique peptide barcode, corresponding to each mRNA sequence, is attached to the expressed protein. These barcodes, cleaved off by a protease and simultaneously quantified by mass spectrometry, reflect the protein expression, enabling a parallel analysis. We validated this method using two mRNAs, each with different untranslated regions (UTRs) but encoding enhanced green fluorescence protein (eGFP), and investigated whether the peptide barcodes could analyze the differential eGFP expression levels. RESULTS: The fluorescence intensity of eGFP, a marker of its expression level, has shown noticeable changes between the two UTR sequences in mRNA-transfected cells when measured using flow cytometry. This suggests alterations in the expression level of eGFP due to the influence of different UTR sequences. Furthermore, the quantified amount of peptide barcodes that were released from eGFP showed consistent patterns with these changes. CONCLUSIONS: The experimental findings suggest that peptide barcodes serve as a valuable tool for assessing protein expression levels. The process of mRNA sequence selection, aimed at maximizing protein expression, can be enhanced by the parallel analysis of peptide barcodes using mass spectrometry.

Analysis of Polypeptides by Amino Acid Analysis.

Amino acid analysis is an accurate method for the composition and quantitation of polypeptides and among these synthetic peptides. Combined with mass spectrometry, it yields a reliable control of peptide quality and quantity prior to conjugation and immunization.Initially peptides are hydrolyzed, preferably in the gas phase, with 6-M HCl at 110 °C for 20-24 h and the resulting amino acids analyzed by chromatography, where the most reliable form is ion exchange chromatography with post-column ninhydrin derivatization. Depending on the hydrolysis conditions, tryptophan is destroyed, and likewise cysteine, unless derivatized, and the amides, glutamine, and asparagine are deamidated to glutamic acid and aspartic acid, respectively. Three different ways of calculating results are suggested, and taking the above limitations into account, a quantitation better than 5% can usually be obtained.

Characterization of Synthetic Peptides by Mass Spectrometry.

In the quality control of synthetic peptides, mass spectroscopy (MS) serves as an optimal method for evaluating authenticity and integrity. Typically, the sequence of a synthetic peptide is already established, thereby directing the focus of analysis towards validating its identity and purity. This chapter outlines straightforward methodologies for conducting MS analyses specifically tailored for synthetic peptides.

Interpretation of Tandem Mass Spectrometry (MS-MS) Spectra for Peptide Analysis.

The aim of this chapter is to give a short introduction to peptide analysis by mass spectrometry (MS) and interpretation of fragment mass spectra. Through examples and guidelines, we will demonstrate how to understand and validate search results and how to perform de novo sequencing based on the often very complex fragmentation pattern obtained by tandem mass spectrometry (also referred to as MSMS). The focus will be on simple rules for interpretation of MSMS spectra of tryptic as well as non-tryptic peptides.

Comparative understanding of peroxide quantitation assays: a case study with peptide drug product degradation.

Peroxide-mediated oxidation of drug molecules is a known challenge faced throughout the pharmaceutical development pathway-from early-stage stability studies to manufacturing processes. During the initial development stage, the major source of peroxide is the formulation excipients, whether they are pre-loaded or generated in situ due to slow degradation, and in the late phase, peroxides can be introduced during sanitization processes or generated via cavitation. In essence, a control strategy for peroxide mitigation often becomes a critical quality attribute for successful drug development. To this end, quantitation of peroxide is essential to monitor the peroxide level to ensure product quality and proposed shelf-life. However, methods for reliable and robust quantitation to detect trace levels of peroxide in a complex drug product matrix become increasingly challenging. This article discusses three high-throughput assays based on absorbance, fluorescence and chemiluminescence measurements to detect peroxide at a low level and compares the methods through validation studies in water. Selected methods have also been tested to understand the forced degradation of model peptide drug products with spiked hydrogen peroxide. Peptide degradation profiles and residual peroxide levels are presented to provide an understanding of the suitability of the quantitation methods and their performance.