Peptidomic Analysis Research Articles

Exploring peptidomes of by-products generated during chhurpi production using Lactobacillus delbrueckii WS4 for identification of novel bioactive peptides.

The considerable value of whey is evident from its significant potential applications and contributions to the functional food and nutraceutical market. The by-products were individually obtained during functional chhurpi and novel soy chhurpi cheese production using defined lactic acid bacterial strains of Sikkim Himalaya's traditional chhurpi. Hydrolysis of substrate proteins by starter proteinases resulted in a comparable peptide content in whey and soy whey which was associated with antioxidant and ACE inhibition potential. Peptidome analysis of Lactobacillus delbrueckii WS4 whey and soy whey revealed the presence of several bioactive peptides including the multifunctional peptides PVVVPPFLQPE and YQEPVLGPVRGPFPIIV. In silico analyses predicted the antihypertensive potential of whey and soy whey peptides with strong binding affinity for ACE active sites. QSAR models predicted the highest ACE inhibition potential (IC50) for the β-casein-derived decapeptide PVRGPFPIIV (0.95 μM) and the Kunitz trypsin inhibitor protein-derived nonapeptide KNKPLVVQF (16.64 μM). Chhurpi whey and soy whey can be explored as a valuable source of diverse and novel bioactive peptides for applications in designer functional foods development.

Intracellular and Extracellular Peptidomes of the Model Plant, Physcomitrium patens.

Here, we report our approach to peptidomic analysis of the plant model Physcomitrium patens. Intracellular and extracellular peptides were extracted under conditions preventing proteolytic digestion by endogenous proteases. The extracts were fractionated on size exclusion columns to isolate intracellular peptides and on reversed-phase cartridges to isolate extracellular peptides, with the isolated peptides subjected to LC-MS/MS analysis. Mass spectrometry data were analyzed for the presence of peptides derived from the known proteins or microproteins encoded by small open reading frames (<100 aa, smORFs) predicted in the moss genome. Experimental details are provided for each step.

Comparative peptidomics analysis reveals the peptides involving tobacco seed ageing

Comparative peptidomics analysis reveals the peptides involving tobacco seed ageing

Peptidomics Unveils Distinct Acetylation Patterns of Histone and Annexin A1 in Differentiated Thyroid Cancer.

Thyroid cancer is a common malignancy of the endocrine system. Nodules are routinely evaluated for malignancy risk by fine needle aspiration biopsy (FNAB), and in cases such as follicular lesions, differential diagnosis between benign and malignant nodules is highly uncertain. Therefore, the discovery of new biomarkers for this disease could be helpful in improving diagnostic accuracy. Thyroid nodule biopsies were subjected to a precipitation step with both the insoluble and supernatant fractions subjected to proteome and peptidome profiling. Proteomic analysis identified annexin A1 as a potential biomarker of thyroid cancer malignancy, with its levels increased in malignant samples. Also upregulated were the acetylated peptides of annexin A1, revealed by the peptidome analysis of the supernatant fraction. In addition, supernatant peptidomic analysis revealed a number of acetylated histone peptides that were significantly elevated in the malignant group, suggesting higher gene transcription activity in malignant tissue. Two of these peptides were found to be robust malignancy predictors, with an area under the receiver operating a characteristic curve (ROC AUC) above 0.95. Thus, this combination of proteomics and peptidomics analyses improved the detection of malignant lesions and also provided new evidence linking thyroid cancer development to heightened transcription activity. This study demonstrates the importance of peptidomic profiling in complementing traditional proteomics approaches.

Identification of bitter peptides in aged Cheddar cheese by crossflow filtration-based Fractionation, Peptidomics, statistical screening and sensory analysis

Despite bitterness being a common flavor attribute of aged cheese linked to casein-derived peptides, excessive bitterness is a sensory flaw that can lead to consumer rejection and economic loss for creameries. Our research employs a unique approach to identify bitter peptides in cheese samples using crossflow filtration-based fractionation, mass spectrometry-based peptidomics, statistics and sensory analysis. Applying peptidomics and statistical screening tools, rather than traditional chemical separation techniques, to identify bitter peptides allows for screening the whole peptide profile. Five peptides—YPFPGP (β-casein [60–65]), YPFPGPIPN (βA2-casein [60–68]), LSQSKVLPVPQKAVPYPQRDMPIQA (β-casein [165–189]), YPFPGPIHNS (βA1-casein [60–69]) and its serine phosphorylated version YPFPGPIHN[S] (βA1-casein [60–69])— demonstrated high levels of bitterness with mean bitterness intensity values above 7 on a 15-point scale. In the future, this data can be combined with the microbial and protease profile of the Cheddar samples to help understand how these factors contribute to bitter taste development.

The secreted peptide BATSP1 promotes thermogenesis in adipocytes

Although brown adipose tissue (BAT) has historically been viewed as a major site for energy dissipation through thermogenesis, its endocrine function has been increasingly recognized. However, the circulating factors in BAT that play a key role in controlling systemic energy homeostasis remain largely unexplored. Here, we performed a peptidomic analysis to profile the extracellular peptides released from human brown adipocytes upon exposure to thermogenic stimuli. Specifically, we identified a secreted peptide that modulates adipocyte thermogenesis in a cell-autonomous manner, and we named it BATSP1. BATSP1 promoted BAT thermogenesis and induced browning of white adipose tissue in vivo, leading to increased energy expenditure under cold stress. BATSP1 treatment in mice prevented high-fat diet-induced obesity and improved glucose tolerance and insulin resistance. Mechanistically, BATSP1 facilitated the nucleocytoplasmic shuttling of forkhead transcription factor 1 (FOXO1) and released its transcriptional inhibition of uncoupling protein 1 (UCP1). Overall, we provide a comprehensive analysis of the human brown adipocyte extracellular peptidome following acute forskolin (FSK) stimulation and identify BATSP1 as a novel regulator of thermogenesis that may offer a potential approach for obesity treatment.

Construction of hypoallergenic microgel by soy major allergen β-conglycinin through enzymatic hydrolysis and lactic acid bacteria fermentation

Soy allergenicity is a public concern, and the combination of multiple processing methods may be a promising strategy for reducing soy allergenicity. In this study, a novel two-step enzymatic hydrolysis followed by lactic acid bacteria fermentation was proposed for the construction of hypoallergenic soybean protein microgel. β-Conglycinin was used as the main soy allergen. The effects of different enzymatic hydrolysis (Alcalase, Neutrase, and Protamex) and LAB fermentation on β-conglycinin microgel formation and its immunoreactivity were investigated. Results showed that the use of different enzymes and the attainment of different degrees of hydrolysis affected the particle distribution and zeta potential in the microgels and leads to differences in microstructure and immunoreactivity. All hydrolysates compared with intact protein accelerated the formation of gel during LAB fermentation. Among the three assayed enzymes, fermented Protamex hydrolysates at 60 min (PF-60) demonstrated a microgel with an overall reduced average particle size (741.20±7.18 nm), lower absolute values of zeta potential (10.43±0.65 mV), and regular gel network. The antigenicity and IgE-binding capacity decreased to the lowest value of 0.30 % and 6.93 %, respectively. Peptidomics and immunoinformatic analysis suggested that PF-60 disrupted 17/30, 16/44, and 23/75 epitopes in the α, α′, and β subunits, respectively. Unlike the LAB-fermented unhydrolyzed β-conglycinin disrupted epitopes mostly located at the loop domain, PF-60 primarily promoted the exposure and disruption of allergen epitopes with β-sheet structure located at the core barrel domain. These findings can provide new perspectives on the preparation of hypoallergenic soybean-gel products on edible particulate systems.

In vitro gastrointestinal digestibility and peptidomic analysis of frankfurters as influenced by different forms of κ-carrageenan

We used peptide profiling to investigate the effects of different forms of κ-carrageenan (a typical hydrocolloid) on the in vitro gastrointestinal digestibility of frankfurters. All incorporated forms of κ-carrageenan were found to decrease the digestibility of meat proteins in the frankfurters, although the effects varied for some peptide fragments. Particle size analysis and super-resolution microscopy revealed that addition of κ-carrageenan in a water suspension resulted in the lowest protein digestibility for frankfurters, followed by κ-carrageenan in a powder form and in a brine suspension. Peptide profiling identified 63 peptides common to all frankfurters after gastrointestinal digestion. In addition, 9 new peptide fragments related to myofibrillar proteins and sarcoplasmic proteins were observed in digestion products from frankfurters that contained κ-carrageenan. Addition of the κ-carrageenan water suspension resulted in a unique pattern of peptide abundance after gastrointestinal digestion of the frankfurters. This pattern was not observed with the powder or brine suspension, both of which contained higher abundances of myosin peptides. However, it is still unclear how different forms of κ-carrageenan caused changes in specific digestion sites, and further investigation is needed to understand this phenomenon. These findings provide novel insights into the effects of various forms of κ-carrageenan on the digestibility of meat proteins in emulsified meat products.

Preventive effect of fermented whey protein mediated by Lactobacillus gasseri IM13 via the PI3K/AKT/FOXO pathway in muscle atrophy

This study investigated the preventive effects of whey protein fermented with Lactobacillus gasseri IM13 (F-WP) against dexamethasone (DEX)-induced muscle atrophy. C2C12 muscle cells were treated with F-WP followed by DEX treatment. Dexamethasone treatment inhibited myotube formation and the expression of myogenic regulatory factors; however, pretreatment with F-WP attenuated DEX-induced damage. The F-WP significantly activated the phosphorylation of the IGF-1/PI3K/AKT pathway and improved muscle homeostasis suppressed by DEX. Moreover, F-WP alleviated the phosphorylation of mTOR, S6K1, and 4E-BP1 and enhanced muscle protein synthesis. Muscle-specific ubiquitin ligases and autophagy lysosomes, which were activated by the dephosphorylation of FOXO3a by DEX treatment, were significantly attenuated by F-WP pretreatment of myotubes. For peptidomic analysis, F-WP was fractionated using preparative HPLC (prep-HPLC), and the AA sequences of 11 peptides were identified using MALDI-TOF/MS/MS. In conclusion, fermentation of whey protein by the specific probiotic strain IM13 produced bioactive peptides with high antioxidant and anti-sarcopenic-sarcopenic effects, which markedly enhanced myogenesis and muscle protein synthesis while diminishing muscle protein degradation compared with intact whey protein.

Application of serum peptidomics for Parkinson's disease in SNCA-A30P mice

Intraneuronal inclusions of alpha-synuclein (α-synuclein, α-syn) are commonly found in the brain of patients with Parkinson's disease (PD). The pathogenesis of the abundant α-syn protein in the blood has been extensively studied to understand its properties better. In recent years, peptidome analysis has received increasing attention. In this study, we identified and analyzed serum peptides from wild-type (WT) and the (Thy-1)-h[A30P] alpha-synuclein transgenic mice (SNCA-A30P mice) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). One thousand eight hundred fifty-six peptides from 771 proteins were analyzed. Among them, 151 peptides from 107 proteins were significantly differentially expressed. The glycoprotein VI platelet pathway (GP6) was the pathway's most significant differentially expressed signaling pathway. Cleavage sites of the differentially expressed peptides may reflect protease distribution and activity. We selected the most significantly differentially expressed peptide, VGGDPI, and found that it contained cathepsin K (Ctsk) and trypsin-1 cleavage sites, suggesting that Ctsk and trypsin-1 may be key peptidases in PD. α-syn is a protein associated with the pathogenesis of PD. mutations in several genes, including SNCA, which encodes α-syn, are associated with the development of PD. Bioinformatics analysis of the physiological pathways related to SNCA genes and apoptosis genes found the five most markedly up-regulated proteins: formin homology 2 domain-containing 1 (FHOD1), insulin receptor substrate 1(IRS1), TRPM8 channel-associated factor 1 (TCAF1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and interleukin-16 (IL-16). Therefore, the differentially expressed peptides in the five precursor protein domains may be potential bioactive peptides associated with α-syn and apoptosis. This study provides a validated peptidomics profile of SNCA-A30P mice and identifies potentially bioactive peptides linked to α-syn and apoptosis.

Peptidomic Analysis and Potential Bioactive Peptides Identification Targeting Endometrial Cancer

Peptidomic Analysis and Potential Bioactive Peptides Identification Targeting Endometrial Cancer

Peptidome analysis reveals critical roles for peptides in a rat model of intestinal ischemia/reperfusion injury.

Intestinal ischemia/reperfusion injury (IIRI) has the potential to be life threatening and is associated with significant morbidity and serious damage to distant sites in the body on account of disruption of the intestinal mucosal barrier. In the present study, we have explored this line of research by comparing and identifying peptides that originated from the intestinal segments of IIRI model rats by using liquid chromatography-mass spectrometry (LC-MS). We also analyzed the basic characteristics, cleavage patterns, and functional domains of differentially expressed peptides (DEPs) between the IIRI model rats and control (sham-operated) rats and identified bioactive peptides that are potentially associated with ischemia reperfusion injury. We also performed bioinformatics analyses in order to identify the biological roles of the DEPs based on their precursor proteins. Enrichment analysis demonstrated the role of several DEPs in impairment of the intestinal mucosal barrier caused by IIRI. Based on the results of comprehensive ingenuity pathway analysis, we identified the DEPs that were significantly correlated with IIRI. We identified a candidate precursor protein (Actg2) and seven of its peptides, and we found that Actg2-6 had a more significant difference in its expression, a longer half-life, and better lipophilicity, hydrophobicity, and stability than the other candidate Actg2 peptides examined. Furthermore, we observed that Actg2-6 might play critical roles in the protection of the intestinal mucosal barrier during IIRI. In summary, our study provides a better understanding of the peptidomics profile of IIRI, and the results indicate that Actg2-6 could be a useful target in the treatment of IIRI.

Peptidomic analysis of follicular fluid in patients with polycystic ovarian syndrome.

Objective: The aim of this study was to analyze and compare the differential expression of peptides within the follicular fluid of polycystic ovary syndrome (PCOS) patients versus normal women by using peptidomics techniques. The underlying mechanisms involved in PCOS pathogenesis will be explored, together with screening and identification of potential functional peptides via bioinformatics analysis. Materials and methods: A total of 12 patients who underwent in vitro fertilization and embryo transfer (IVF-ET) at the Reproductive Medicine Center of Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine from 1 September 2022 to 1 November 2022 were included in this study. The follicular fluid of PCOS patients (n = 6) and normal women (n = 6) were collected. The presence and concentration differences of various peptides were detected by the LC-MS/MS method. GO and KEGG analysis were performed on the precursor proteins of the differentially-expressed peptides, and protein network interaction analysis was carried out to identify functionally-relevant peptides among the various peptides. Results: A variety of peptides within the follicular fluid of PCOS versus normal patients were detected by peptidomics techniques. Altogether, 843 upregulated peptides and 236 downregulated peptides were detected (absolute fold change ≥2 and p < 0.05). Of these, 718 (718 = 488 + 230) peptides were only detected in the PCOS group, while 205 (205 = 174 + 31) were only detected in the control group. Gene Ontology enrichment and pathway analysis were performed to characterize peptides through their precursor proteins. We identified 18 peptides from 7 precursor proteins associated with PCOS, and 4 peptide sequences were located in the functional domains of their corresponding precursor proteins. Conclusion: In this study, differences in the follicular development of PCOS versus normal patients were revealed from the polypeptidomics of follicular development, which thus provided new insights for future studies on the pathological mechanisms of PCOS development.

Impact of Wooden Breast myopathy on in vitro protein digestibility, metabolomic profile, and cell cytotoxicity of cooked chicken breast meat

This study investigated the impacts of Wooden Breast (WB) abnormality on in vitro protein digestibility and cytotoxicity of cooked chicken breast meat. Chicken breasts without (non-WB, n = 6) or with severe WB condition (WB, n = 6) were cooked and subjected to static in vitro protein digestion. The results showed no significant differences in free-NH2, degree of hydrolysis and distribution of peptide molecular weight between non-WB and WB samples at late intestinal digestion (P5), suggesting no adverse effects of WB on protein digestibility. Based on peptidomic analysis, P5 fraction of WB showed greater content of peptides with oxidative modification than that of non-WB. Untargeted metabolomics did not find any metabolites with potential toxicity either in non-WB and WB. Hydrolyzed non-WB and WB (1.56 to 100 µg/mL) did not affect viability of Caco-2 and Vero cells but addition of WB samples reduced Caco-2 cell viability compared with non-WB.

A novel alcalase-hydrolyzed soybean meal hydrolysates prepared using by-product material: Structure, function property, sensory property, and biological activity

This work investigated the effects of different hydrolysis times (0.5, 1 and 2 h) on the structure, interface and sensory properties, and biological activity of soybean meal peptides obtained by alcalase protease hydrolysis. After hydrolyzation, the molecular weight of soybean meal hydrolysates (SMHs) was below 10 kDa. An increase in the number of β-turns and a decrease in α-helices suggested the unfolding of soybean meal proteins. The ultraviolet absorption and fluorescence intensities of SMHs increased, suggesting an increase in the content of free amino acids, which likely contributed to the biological activity of SMHs. Additionally, after hydrolyzation for 1 h, the SMHs showed the highest antioxidant and ACE inhibitory activity. LC-MS/MS-based peptidomics analysis showed 101 differential peptides in hydrolysates among different hydrolysis times with reliable potential biological activities. However, the emulsification of the SMHs decreased due to the exposure of hydrophilic amino acids. All in all, hydrolysis time is a factor that influences the peptide sequence and properties, resulting in different antioxidant and ACE inhibitory activities.

Effects of processing on component interactions and peptide profiles of preterm infant formula based on peptidomics by Q Exactive plus analysis

The study aimed to investigate the effects of processing on the component interactions, the peptide profile as well as the bioactive peptides of preterm infant formula after digestion through EASY-nLC1200 Q Exactive Plus system. Results indicated that whey proteins and caseins transferred from serum and pellet fractions to fat fraction to recombine the milk fat globule membrane destroyed after homogenization. Protein interactions caused the changes of nitrogen distribution, protein profiles, and morphology after pasteurization and spray drying. Peptidomics analysis demonstrated that the component interactions of preterm infant formula affected the peptide profiles after digestion shown by the different peptide intensities of certain amino acid compositions and the totally different abundance of the peptides in heatmap. The hydrolysates of five groups exhibited 334, 274, 421, 214, and 265 unique peptides belonging to 144, 116, 185, 101, and 231 master accession proteins respectively. The top 8 abundant proteins identified were β-casein, Lipocln, αS1-casein, αS2-casein, κ-casein, bovine Glycosylation-dependent cell adhesion molecule, α-lactalbumin, and Albumin. The number of bioactive peptides increased after processing but were different from each other. In total, component interactions during processing of preterm infant formula could change the peptide profile and bioactive peptides after in vitro digestion.

The molecular mechanisms of extracellular matrix-derived hydrogel therapy in idiopathic pulmonary fibrosis models

Idiopathic Pulmonary Fibrosis (IPF) is a progressively debilitating lung condition characterized by oxidative stress, cell phenotype shifts, and excessive extracellular matrix (ECM) deposition. Recent studies have shown promising results using decellularized ECM-derived hydrogels produced through pepsin digestion in various lung injury models and even a human clinical trial for myocardial infarction. This study aimed to characterize the composition of ECM-derived hydrogels, assess their potential to prevent fibrosis in bleomycin-induced IPF models, and unravel their underlying molecular mechanisms of action. Porcine lungs were decellularized and pepsin-digested for 48 h. The hydrogel production process, including visualization of protein molecular weight distribution and hydrogel gelation, was characterized. Peptidomics analysis of ECM-derived hydrogel contained peptides from 224 proteins. Probable bioactive and cell-penetrating peptides, including collagen IV, laminin beta 2, and actin alpha 1, were identified. ECM-derived hydrogel treatment was administered as an early intervention to prevent fibrosis advancement in rat models of bleomycin-induced pulmonary fibrosis. ECM-derived hydrogel concentrations of 1 mg/mL and 2 mg/mL showed subtle but noticeable effects on reducing lung inflammation, oxidative damage, and protein markers related to fibrosis (e.g., alpha-smooth muscle actin, collagen I). Moreover, distinct changes were observed in macroscopic appearance, alveolar structure, collagen deposition, and protein expression between lungs that received ECM-derived hydrogel and control fibrotic lungs. Proteomic analyses revealed significant protein and gene expression changes related to cellular processes, pathways, and components involved in tissue remodeling, inflammation, and cytoskeleton regulation. RNA sequencing highlighted differentially expressed genes associated with various cellular processes, such as tissue remodeling, hormone secretion, cell chemotaxis, and cytoskeleton engagement. This study suggests that ECM-derived hydrogel treatment influence pathways associated with tissue repair, inflammation regulation, cytoskeleton reorganization, and cellular response to injury, potentially offering therapeutic benefits in preventing or mitigating lung fibrosis.

Peptidomics Analysis of Soy Protein Hydrolysates-Antioxidant Properties and Mechanism of their Inhibition of the Oxidation of Palm Olein during Frying Cycles.

This study determined for the first time the structure of the peptides (i.e., peptidomics) in soy protein hydrolysates and elucidated their effects on an oil's oxidative stability during frying cycles. The oil investigated was palm olein during 0, 4, 8, and 12 frying cycles of plantain banana chips. Proteins were extracted and hydrolyzed with two proteases. Trypsin hydrolysate (HTRY) exhibited higher anti-radical activity (DPPH, 70.2%) than the control (unhydrolyzed proteins, 33.49%) and pepsin hydrolysate (HPEP, 46.1%) at 200 µg/mL. HPEP however showed a 4.6-fold greater reduction of ferric ions (FRAP) while also possessing a higher peroxyl radical scavenging ability (716 ± 30 µM Trolox Eq/g) than HTRY (38.5 ± 35 µM Trolox Eq/g). During oil oxidative stability tests, HPEP improved the oxidative stability of the palm olein oil after 8 and 12 frying cycles, characterized by lower concentrations of hydroperoxides, and carbonyl and volatile compounds. HTRY however exerteda pro-oxidant activity. Structural data from SDS-PAGE and tandem mass spectrometry showed that the mechanism for the greater activity of the pepsin hydrolysate occurred due to unique structural features and a higher percentage of short-chain peptides. This was justified by a 25, 31, and 48% higher contents of tryptophan, histidine, and methionine, respectively (important amino acids with hydrogen atom transfer and electron-donating capacities) in the peptides identified in the pepsin hydrolysate.

Subclinical and clinical acute kidney injury share similar urinary peptide signatures and prognosis.

Acute kidney injury (AKI) is a frequent and severe condition in intensive care units (ICUs). In 2020, the Acute Dialysis Quality Initiative (ADQI) group proposed a new stage of AKI, referred to as stage 1S, which represents subclinical disease (sAKI) defined as a positive biomarker but no increase in serum creatinine (sCr). This study aimed to determine and compare the urinary peptide signature of sAKI as defined by biomarkers. This is an ancillary analysis of the prospective, observational, multinational FROG-ICU cohort study. AKI was defined according to the Kidney Disease Improving Global Outcome definition (AKIKDIGO). sAKI was defined based on the levels of the following biomarkers, which exceeded the median value: neutrophil gelatinase-associated lipocalin (pNGAL, uNGAL), cystatin C (pCysC, uCysC), proenkephalin A 119-159 (pPENKID) and liver fatty acid binding protein (uLFABP). Urinary peptidomics analysis was performed using capillary electrophoresis-mass spectrometry. Samples were collected at the time of study inclusion. One thousand eight hundred eighty-five patients had all biomarkers measured at inclusion, which included 1154 patients without AKI (non-AKIKDIGO subgroup). The non-AKIKDIGO subgroup consisted of individuals at a median age of 60 years [48, 71], among whom 321 (27.8%) died. The urinary peptide signatures of sAKI, regardless of the biomarkers used for its definition, were similar to the urinary peptide signatures of AKIKDIGO (inflammation, haemolysis, and endothelial dysfunction). These signatures were also associated with 1-year mortality. Biomarker-defined sAKI is a common and severe condition observed in patients within intensive care units with a urinary peptide signature that is similar to that of AKI, along with a comparable prognosis.

Identification of an Antimicrobial Peptide from the Venom of the Trinidad Thick-Tailed Scorpion Tityus trinitatis with Potent Activity against ESKAPE Pathogens and Clostridioides difficile.

Envenomation by the Trinidad thick-tailed scorpion Tityus trinitatis may result in fatal myocarditis and there is a high incidence of acute pancreatitis among survivors. Peptidomic analysis (reversed-phase HPLC followed by MALDI-TOF mass spectrometry and automated Edman degradation) of T. trinitatis venom led to the isolation and characterization of three peptides with antimicrobial activity. Their primary structures were established asTtAP-1 (FLGSLFSIGSKLLPGVFKLFSRKKQ.NH2), TtAP-2 (IFGMIPGLIGGLISAFK.NH2) and TtAP-3 (FFSLIPSLIGGLVSAIK.NH2). In addition, potassium channel and sodium channel toxins, present in the venom in high abundance, were identified by CID-MS/MS sequence analysis. TtAP-1 was the most potent against a range of clinically relevant Gram-positive and Gram-negative aerobes and against the anaerobe Clostridioides difficile (MIC = 3.1-12.5 µg/mL). At a concentration of 1× MIC, TtAP-1 produced rapid cell death (<15 min against Acinetobacter baumannii and Staphylococcus aureus). The therapeutic potential of TtAP-1 as an anti-infective agent is limited by its high hemolytic activity (LC50 = 18 µg/mL against mouse erythrocytes) but the peptide constitutes a template for the design of analogs that maintain the high bactericidal activity against ESKAPE pathogens but are less toxic to human cells. It is suggested that the antimicrobial peptides in the scorpion venom facilitate the action of the neurotoxins by increasing the membrane permeability of cells from either prey or predator.