Pilot study of incidence of gastroesophageal reflux after lung resection.

Patients undergoing lung resection may be at risk of gastroesophageal reflux (GER) and silent aspiration following surgery. Defining high-risk patients may lead to prevention strategies for silent aspiration and subsequent exacerbation of underlying pulmonary disease. A pilot study of 50 patients was performed to investigate postoperative gastroesophageal reflux disease (GERD) symptoms and the pepsin concentration in saliva. Patients answered a questionnaire concerning GERD symptoms before lung surgery and at the time of discharge. Saliva samples were obtained before surgery, on the third postoperative day and at discharge. Pepsin concentration was measured with Peptest. The pepsin concentration in saliva following resection was significantly elevated on postoperative day 3, but it returned to the baseline level at discharge. Patients undergoing resection of four or more lung subsegments had a continuously elevated pepsin concentration in saliva on postoperative day 3 [mean difference 65.63 ng/mL, 95% confidence interval (CI): 9.130-122.1] and at discharge (mean difference 76.22 ng/mL, 95% CI: 19.72-132.7). Patients with a >10% reduction of forced expiration volume in one second also had a continuous elevated pepsin concentration from the 3rd postoperative day. Lung resection resulted in elevated pepsin concentration in the saliva, which persisted in patients who received resections equivalent to or more than right middle lobectomy in volume. Resection of large volumes of lung may lead to anatomical changes and changes in breathing patterns and result in GER.

Does Extraesophageal Reflux Support the Development of Lung Adenocarcinoma? Analysis of Pepsin in Bronchoalveolar Lavage in Non-Smoker Patients.

The significance of extraesophageal reflux as a risk factor in lung adenocarcinoma has been understudied. In this study, we investigated whether extraesophageal reflux leads to higher pepsin concentrations in bronchoalveolar lavage (BAL) in patients with lung adenocarcinoma compared to controls. Subjects were recruited from non-smoker patients (lifelong non-smokers and ex-smokers with more than 5 years of non-smoking history) who had undergone bronchoscopy due to pulmonary abnormalities on a CT scan and met the inclusion criteria. Based on histological verification of the lung process, the patients were divided into three groups: (1) lung adenocarcinoma, (2) pulmonary metastases, and (3) lung sarcoidosis. Lung adenocarcinoma cases were further categorized as central or peripheral. BAL samples collected during bronchoscopy were quantitatively analyzed by enzyme-linked immunosorbent assay (ELISA) to measure pepsin levels. No statistically significant difference in pepsin concentration was observed between the lung adenocarcinoma group and control groups (p = 0.135). After excluding hemorrhagic BAL samples, the pepsin concentration was significantly the lowest in patients with lung adenocarcinoma (p = 0.023) compared to the control groups. The results of the study do not support the hypothesis of a higher occurrence of extraesophageal reflux (evaluated as the amount of pepsin in BAL) in non-smoker patients with lung adenocarcinoma.

Screening of Organic Acid Type and Dosage in Drinking Water for Young Rabbits.

Organic acids (OAs) are employed in animal feed to regulate gastrointestinal disorders and diarrhoea thanks to their ability to modulate the gastrointestinal environment and their antimicrobial capacity. However, there is not enough evidence regarding the most adequate OA and its effectiveness in rabbit farming. Therefore, the aim of this study was to screen and evaluate the response of young rabbits to six OAs, administered via drinking water, at three different concentrations (pH levels). Organic acids (acetic, ACET; formic, FOR; propionic, PROP; lactic, LAC; citric, CIT; and butyric, BUT) were tested at three concentrations (pH 3, 4, and 5). A negative control (CON; non-acidified water) was also included. We used 240 weaned rabbits (28 days old) divided into 2 batches. In each batch, animals were randomly allocated to 1 of the 19 experimental treatments and were housed in group cages of 6 animals per cage, treatment, and batch. Among the 240 rabbits, an additional cage with 6 animals was included to determine the initial physiological state of the animals. All animals were fed with commercial pelleted feed throughout the whole experiment. The duration of the study was 7 days, until 35 days of age. At 31 and 35 days of age, in each batch, three animals per day and treatment were slaughtered. The pH of the digestive contents in the fundus, antrum, duodenum, jejunum, ileum, and cecum, as well as the gastric pepsin enzyme activity, was measured. Water and feed consumption per cage and individual body weight (BW) were recorded daily. The type and dosage of OAs affected water intake. ACET 3, PROP 3, and BUT 3 reduced water intake compared to CON, negatively impacting feed intake and weight gain. FOR and CIT acids led to the highest BW and weight gain at 35 days, compared to PROP, LAC, and BUT (p < 0.05); showing ACET intermediate values. While OAs had limited effects on gastric and small intestine pH, acidified water at pH 4 and 5 lowered ileum and caecum pH (p < 0.05) compared to pH 3. Acidified water at pH 4 showed the highest (p < 0.05) pepsin activity compared to pH 3 and pH 5. Considering the limited sample size and short-term assessment period of our screening test, the OAs with the highest potential for use in post-weaning rabbits were FOR, ACET, and CIT at pH 4. The selected combinations did not exhibit any early adverse effects in young rabbits. These results should be further confirmed in a broader population of animals. It would also be advisable to extend the application of OAs over longer periods to evaluate their effects throughout the entire growing period of rabbits.

Structural Catalytic Core of the Members of the Superfamily of Acid Proteases.

The superfamily of acid proteases has two catalytic aspartates for proteolysis of their peptide substrates. Here, we show a minimal structural scaffold, the structural catalytic core (SCC), which is conserved within each family of acid proteases, but varies between families, and thus can serve as a structural marker of four individual protease families. The SCC is a dimer of several structural blocks, such as the DD-link, D-loop, and G-loop, around two catalytic aspartates in each protease subunit or an individual chain. A dimer made of two (D-loop + DD-link) structural elements makes a DD-zone, and the D-loop + G-loop combination makes a psi-loop. These structural markers are useful for protein comparison, structure identification, protein family separation, and protein engineering.

In vitro model to evaluate effect of acidic pepsin on vocal fold barrier function.

The pathophysiology of laryngopharyngeal reflux (LPR) and its impact on the vocal fold is not well understood, but may involve acid damage to vocal fold barrier functions. Two different components encompass vocal fold barrier function: the mucus barrier and tight junctions. Mucus retained on epithelial microprojections protects the inside of the vocal fold by neutralizing acidic damage. Tight junctions control permeability between cells. Here we developed an in vitro experimental system to evaluate acidic injury and repair of vocal fold barrier functions. We first established an in vitro model of rat vocal fold epithelium that could survive at least one week after barrier function maturation. The model enabled repeated evaluation of the course of vocal fold repair processes. Then, an injury experiment was conducted in which vocal fold cells were exposed to a 5-min treatment with acidic pepsin that injured tight junctions and cell surface microprojections. Both of them healed within one day of injury. Comparing vocal fold cells treated with acid alone with cells treated with acidic pepsin showed that acidic pepsin had a stronger effect on intercellular permeability than acid alone, whereas pepsin had little effect on microprojections. This result suggests that the proteolytic action of pepsin has a larger effect on protein-based tight junctions than on phospholipids in microprojections. This experimental system could contribute to a better understanding of vocal fold repair processes after chemical or physical injuries, as well as voice problems due to LPR pathogenesis.

Acidic Pepsin Affects Laryngeal Carcinoma Cell Growth and Invasion Through Glycolysis.

OBJECTIVE: The pathogenic mechanism underlying the effects of acidic pepsin in laryngeal cancer remains unclear. This study investigated whether acidic pepsin influences Glut-1 expression and glycolytic activity in laryngeal carcinoma cells and whether it plays a role in the growth and migration of these cells through glycolysis. STUDY DESIGN: In vitro study. SETTING: A university-affiliated hospital. METHODS: Laryngeal carcinoma TU 212 and TU 686 cells were treated with acidic pepsin and 2-deoxy-d-glucose (2-DG), then transfected with Glut-1 small interfering RNA (siRNA). Glucose uptake was detected by a radioimmunoassay counter, lactate secretion was detected by a lactic acid kit, and Glut-1 expression was detected by western blotting. Cell viability, migration and invasion, and clonal formation were assessed using the Cell Counting Kit-8, Transwell chamber, and clonal formation assays, respectively. RESULTS: Acidic pepsin significantly increased Glut-1 expression in laryngeal carcinoma cells compared with the control group (P < .01). It also significantly enhanced 18F-fluorodeoxyglucose (Cin/Cout) uptake, lactate secretion, cell viability, migration, invasion, and clonal formation in laryngeal carcinoma cells compared with the control group (P < .01). The glycolytic inhibitor 2-DG and Glut-1 siRNA significantly reversed the effects of acidic pepsin on laryngeal carcinoma cells (P < .01). CONCLUSION: Acidic pepsin enhances the growth and migration of laryngeal carcinoma cells by upregulating Glut-1, thus promoting glycolysis.

Construction of Metal-Organic Framework-Based Heterogeneous Pepsin and Its Degradation Performance and Mechanism for Phthalic Acid Esters.

Biological enzyme-driven degradation of environmental pollutants has attracted widespread attention because it is ecofriendly and highly efficient. Immobilized enzyme technology has emerged as a promising technique in enzymology that addresses the limitations associated with free enzymes. Traditional solid-loaded enzyme substrates are often affected by blockages and restricted substrate accessibility. In this study, we synthesized an efficient heterogeneous pepsin catalyst, named PEP@M-MIL100(Fe), by covalently combining carboxylated ferrite structural expanded metal-organic frameworks with pepsin. This catalyst demonstrated excellent environmental adaptability and remarkable catalytic degradation capabilities. Notably, it rapidly degraded the persistent microplastic pollutant diisononyl phthalate (DINP) within just 150 min, with a removal efficiency of up to 95.88%. Impressively, even after 10 consecutive uses, the catalyst maintained its high performance. We proposed an innovative steady-state heterogeneous enzyme-catalyzed degradation mechanism, i.e., diffusion (D)-absorption (A)-binding (B)-reaction (R)-degradation (D)-link mechanism, which emphasizes the influence of substrate diffusion rates in this process. This work presents the first successful application of pepsin to DINP degradation and offers a sustainable and effective approach for addressing contemporary pollution challenges.

How D-amino acids embedded in the protein sequence modify its digestibility: Behaviour of digestive enzymes tested on a model peptide used as target.

D-amino acids can affect the action of digestive enzymes, hence the protein digestion. In this work the behaviour of the main stomach and gut digestive enzymes (pepsin, trypsin, and chymotrypsin) in the presence of D-amino acids in the protein chain was monitored over time using a model peptide, Ac-LDAQSAPLRVYVE-NH2 (belonging to ß-lactoglobulin, position 48-60), where L-amino acids were systematically substituted by D-amino acids. The results showed several changes in the behaviour of digestive enzymes, not only when the D-amino acids are inserted at the specific cleavage sites (after Val-57), but in some cases also when in distant positions. The effect seemed more pronounced in the case of pepsin rather than the gut enzymes, possibly indicating a better resilience of the upper gut phase of digestion to racemization. These results demonstrated that racemization could impair nutritional value by slowing down digestibility and has different effects according to the enzyme/amino acids involved.

Quantifying LAGA mutated mouse IgG2a monoclonal antibody with a rapid pepsin digestion enabled immunoaffinity LC/MS/MS assay.

A sensitive and specific bioanalytical method was required to measure the exposure of a LAGA-mutated surrogate mouse IgG2a monoclonal antibody in mouse plasma, but the lack of highly specific reagents for the LAGA mutant hindered the development of a ligand-binding assay. Equally problematic is that no sensitive unique tryptic peptides suitable for quantitative mass spectrometric analysis could be identified in the mIgG2a complementarity-determining regions. To overcome these challenges, a trypsin alternative pepsin, an aspartic protease, was systematically investigated for its use in digesting the mutated mIgG2a antibody to allow generation of signature peptides for the bioanalytical quantification purpose. After a series of evaluations, a rapid one-hour pepsin digestion protocol was established for the mutated Fc backbone. Consequently, a new pepsin digestion-based liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was successfully developed to support the mouse pharmacokinetic (PK) sample analysis. In brief, robust and reproducible C-terminal cleavage of both leucine and phenylalanine near the double mutation site of the mutated mIgG2a was accomplished at pH ≤2 and 37°C. Combined with a commercially available rat anti-mIgG2a heavy-chain antibody, the established immunoaffinity LC/MS/MS assay achieved a limit of quantitation of 20 ng/mL in the dynamic range of interest with satisfactory assay precision and accuracy. The successful implementation of this novel approach in discovery PK studies eliminates the need for tedious and costly generation of specific immunocapturing reagents for the LAGA mutants. The approach should be widely applicable for developing popular LAGA mutant-based biological therapeutics.

Protective Encapsulation of a Bioactive Compound in Starch-Polyethylene Glycol-Modified Microparticles: Degradation Analysis with Enzymes.

Starch is a promising polymer for creating novel microparticulate systems with superior biocompatibility and controlled drug delivery capabilities. In this study, we synthesized polyethylene glycol (PEG)-modified starch microparticles and encapsulated folic acid using a solvent-mediated acid-base precipitation method with magnetic stirring, which is a simple and effective method. To evaluate particle degradation, we simulated physiological conditions by employing an enzymatic degradation approach. Our results with FTIR and SEM confirmed the successful synthesis of starch-PEG microparticles encapsulating folic acid. The average size of starch microparticles encapsulating folic acid was 4.97 µm and increased to 6.01 µm upon modification with PEG. The microparticles were first exposed to amylase at pH 6.7 and pepsin at pH 1.5 at different incubation times at physiological temperature with shaking. Post-degradation analysis revealed changes in particle size and morphology, indicating effective enzymatic degradation. FTIR spectroscopy was used to assess the chemical composition before and after degradation. The initial FTIR spectra displayed characteristic peaks of starch, PEG, and folic acid, which showed decreased intensities after enzymatic degradation, suggesting alterations in chemical composition. These findings demonstrate the ongoing development of starch-PEG microparticles for controlled drug delivery and other biomedical applications and provide the basis for further exploration of PEG-starch as a versatile biomaterial for encapsulating bioactive compounds.

Enhanced modified poly-tyrosine voltammetric sensor for the quantification detection of salivary pepsin.

Pepsin as an aspartic acid protease member and one of the three foremost proteolytic enzymes in the digestive system is essential to be detected. An electrochemically polymerized tyrosine film on carbon paste electrode (pTyr/CPE) has been synthesized by electro-polymerization donating an affordable electrochemical sensor to sense salivary pepsin as a diagnostic technique for gastroesophageal reflux disease (GRD) due to saliva collection is non-invasive and relatively comfortable. The pTyr/CPE was applied for Voltammetric sensing of pepsin and its quantification in phosphate buffer solution of pH 2.0 (PBS). Scanning electron microscopy (SEM) was conducted to learn the surface morphology. Cyclic voltammetry (CV), differential pulse voltammetry (DPVs), chronoamperometry (CA), and electrochemical impedance spectroscopy (EIS) were developed to realize the electrocatalytic activity of the sensor. The pTyr/CPE proceeded as a sensitive detector to pepsin with two linear ranges from 1 to 20 & 20 to 100 ng/mL donating two limits of detection as 0.5 & 0.09 ng/mL, respectively, and high selectivity toward pepsin, as well as stability and fast response of 1.5 s. Consequently, it is guessed that the pTyr/CPE sensor could be supportive for the initial diagnosis of GRD through the detection of pepsin in saliva. Finally, we quantified the pepsin levels in saliva samples of LPR patients (n = 2), showing that the results were agreeable with those from the electrochemical sensor.

Identification and Characterization of a Pepsin- and Chymotrypsin-Resistant Peptide in the α Subunit of the 11S Globulin Legumin from Common Bean (Phaseolus vulgaris L.).

The 11S globulin legumin typically accounts for approximately 3% of the total protein in common beans (Phaseolus vulgaris). It was previously reported that a legumin peptide of approximately 20 kDa is resistant to pepsin digestion. Sequence prediction suggested that the pepsin-resistant peptide is located at the C-terminal end of the α-subunit, within a glutamic acid-rich domain, overlapping with a chymotrypsin-resistant peptide. Using purified legumin, the peptide of approximately 20 kDa was found to be resistant to pepsin digestion in a pH-dependent manner, and its location was determined by two-dimensional gel electrophoresis and LC-MS-MS. The location of the chymotrypsin-resistant peptide was confirmed by immunoblotting with peptide-specific polyclonal antibodies. The presence of a consensus site for proline hydroxylation and arabinosylation, the detection of hydroxyproline residues, purification by lectin affinity chromatography, and a difference in electrophoretic migration between the chymotrypsin- and pepsin-resistant peptides suggest the presence of a large O-glycan within these peptides.

Impact of quercetin conjugation using alkaline and free radical methods with tandem ultrasonication on the functional properties of camel whey and its hydrolysates.

The structural and functional properties of whey-quercetin and whey hydrolysate-quercetin conjugates synthesized using alkaline and free radical-mediated methods (AM and FRM) coupled with sonication were studied. FTIR showed new peaks at 3000-3500 cm-1 (N-H stretching regions) and the 1000-1100 cm-1 region with the conjugates. Conjugation increased the random coils and α-helix content while decreasing the ß-sheets and turns. It also increased the particle size and surface hydrophobicity which was significantly (p < 0.05) higher in AM than FRM conjugates. AM conjugates had higher radical scavenging activity but lower quercetin content than FRM conjugates. Overall, the functional properties of whey-quercetin conjugates were better than whey hydrolysate-quercetin conjugates. However, hydrolysate conjugates had significantly higher denaturation temperatures irrespective of the method of production. Sonication improved the radical scavenging activity and quercetin content of FRM conjugates while it decreased both for AM conjugates. This study suggested that whey-quercetin conjugates generally had better quality than whey hydrolysate conjugates and sonication tended to further improve these properties. This study highlights the potential for using camel whey or whey hydrolysate-quercetin conjugates to enhance the functional properties of food products in the food industry.

Use of Betaine HCl with Pepsin in Esophageal Cancer Patient: A Case Report.

The principal mechanisms surrounding gastrointestinal (GI) side effects due to chemotherapy are unclear, whereas the information regarding symptom management of patients with esophageal cancer post-esophagectomy is lacking. Esophagectomy patients are left with significant anatomical changes to the GI tract, including the cutting of the vagus nerve, which regulates gastric secretions, gastric acid pH, and motility. A 76-year-old male patient self-referred himself to the clinical dietitian for nutritional management of chronic nausea, fatigue, weight loss, and dumping syndrome 9 months post-esophagectomy, which was not responsive to medications. A physical functional nutritional assessment with evaluation of diet history and elimination suggested gastric hypochlorhydria. Gastric acid is needed for the active absorption of iron, zinc, B complex vitamins, especially B12, and digestion of consumed proteins. A digestive supplement, betaine hydrochloric acid with pepsin (BHClP), was introduced, and the patient ingested 1 capsule containing 500 mg betaine hydrochloride and 23.5 mg pepsin prior to protein-containing meals and reported a substantial decrease in GI symptoms while eating a regular diet with no limitations. He gained necessary weight and energy for daily activities. After a few months, the patient discontinued BHClP, and GI symptoms and dumping syndrome returned, leading to a loss of 7.5% of his body weight. The patient reinitiated the supplement and GI symptoms dissipated, and weight was restored. BHClP provided metabolic therapeutic benefit to optimize the patient's oral intake, preventing further complications and malnutrition. The success with BHClP for this patient case suggests that more research is needed to fully realize the mechanisms and clinical usage.

Pepsin immobilization on activated carbon and functionalized with glutaraldehyde and genipin for the synthesis of antioxidant peptides of goat casein.

In this article, the synthesis of antioxidant peptides in the enzymatic hydrolysis of caprine casein was analyzed at three different time points (60 min, 90 min, and 120 min) using immobilized pepsin on activated and modified carbon (AC, ACF, ACG 50, ACG 100). The immobilization assays revealed a reduction in the biocatalysts' activity compared to the free enzyme. Among the modified ones, ACG 50 exhibited greater activity and better efficiency for reuse cycles, with superior values after 60 min and 90 min. Peptide synthesis was observed under all studied conditions. Analyses (DPPH, ß-carotene/linoleic acid, FRAP) confirmed the antioxidant potential of the peptides generated by the immobilized enzyme. However, the immobilized enzyme in ACG 50 and ACG 100, combined with longer hydrolysis times, allowed the formation of peptides with an antioxidant capacity greater than or equivalent to those generated by the free enzyme, despite reduced enzymatic activity.

Relationship between microstructure formation and in vitro starch digestibility in baked gluten-starch matrices.

Increased prevalence of diabetes prompts the development of foods with reduced starch digestibility. This study analyzed the impact of adding soluble dietary fiber (inulin-IN; polydextrose-PD) to baked gluten-starch matrices (7.5-13%) on microstructure formation and in vitro starch digestibility. IN and PD enhanced water-holding capacity, the hardness of baked matrices, and lowered water activity in the formulated matrices, potentially explaining the reduced starch gelatinization degree as IN or PD concentration increased. A maximum gelatinization decrease (26%) occurred in formulations with 13% IN. Micro-CT analysis showed a reduction in total and open porosity, which, along with the lower gelatinization degree, may account for the reduced in vitro starch digestibility. Samples with 13% IN exhibited a significantly lower rapidly available glucose fraction (8.56 g/100 g) and higher unavailable glucose fraction (87.76 g/100 g) compared to the control (34.85 g/100 g and 47.59 g/100 g, respectively). These findings suggest the potential for developing healthier, starch-rich baked foods with a reduced glycemic impact.

Interactions between Salivary Proteins and Apple Polyphenols and the Fate of Complexes during Gastric Digestion.

Beneficial polyphenols in apples can reach the stomach as complexes formed with salivary proteins. The present study aimed at documenting the interactions between salivary proteins and cider apple polyphenols and the fate of complexes during gastric digestion. A polyphenolic extract was mixed with human saliva, and interactions were characterized by analyzing proteins and polyphenols in the insoluble and soluble fractions of the mixtures, before and after in vitro gastric digestion. Results confirmed that proline-rich proteins can efficiently precipitate polyphenols and suggested that two zinc-binding proteins can also form insoluble complexes with polyphenols. The classes of polyphenols involved in such complexes depended on the polyphenol-to-protein ratio. In vitro gastric digestion led to extensive proteolysis of salivary proteins, and we formulate the hypothesis that the resulting peptides can interact with and precipitate some procyanidins. Saliva may therefore partly modulate the bioaccessibility of at least procyanidins in the gastric compartment.

Elevated Saliva Pepsin Concentration as a Risk Factor for Asthma in Children with Allergic Rhinitis: A Preliminary Study.

Objective: This study aimed to explore whether saliva pepsin concentration (SPC) could be regarded as a risk factor for the occurrence and unfavorable control of asthma in children with allergic rhinitis. Methods: A prospective study was conducted on a group of 20 consecutive children newly diagnosed with allergic rhinitis and asthma (referred to as the asthma group). All these children underwent fractional exhaled nitric oxide (FeNO) measurement, lung function tests, and assessment of asthma control using the 7-item Childhood Asthma Control Test (C-ACT) score. Simultaneously, a control group consisting of 20 children with simple allergic rhinitis, matched for baseline characteristics, was included. SPC measurement was performed in the two groups. Results: The SPC value was significantly higher in the asthma group than that in the control group (165.0 ± 82.8 ng/mL vs 68.4 ± 34.5 ng/mL) (P < 0.001). In the asthma group, SPC was independently associated with FeNO, the ratio of forced expiratory volume in 1 second (FEV1) to forced vital capacity (FVC), and forced expiratory flow at 50% and 75% of FVC (FEF50 and FEF75) (all P < 0.05). The severity of nasal symptoms evaluated by the visual analogue scale (N-VAS) was independently associated with FEF75, the maximal mid-expiratory flow (MMEF), and C-ACT score (P < 0.05). Conclusion: Direct pepsin exposure and uncontrolled nasal symptoms may play crucial roles in the pathogenesis and progression of childhood allergic asthma. The SPC value can be considered as a risk factor for asthma in children with allergic rhinitis.

Analysis of Protein Digestion and Absorption Using a Ussing Chamber to Simulate the Environment in the Digestive Tract.

The Ussing chamber is a tool for analyzing drug absorption. We investigated whether the Ussing chamber can be used to analyze the process from digestion to absorption of protein in the gastrointestinal tract. Mixtures containing infant formula, whole cow's milk, processed soy milk, enteral nutrition, or human breast milk, were placed in the apical membrane side equipped with Caco-2 cells. After the addition of first pepsin then pancreatin, samples from the apical and basal membranes were collected. Infant formula showed the highest digestibility and absorption rate. This may be attributed to the presence of whey protein, which is rapidly digested and absorbed. The digestion and absorption of human breast milk showed different results in each donor, suggesting that digestion and absorption may vary among individuals. We concluded that the Ussing chamber can continuously analyze the process from digestion to absorption of proteins in the gastrointestinal tract.

Accelerating Proteomics Using Broad Specificity Proteases.

Trypsin is the gold-standard protease in bottom-up proteomics, but many sequence stretches of the proteome are inaccessible to trypsin and standard LC-MS approaches. Thus, multienzyme strategies are used to maximize sequence coverage in post-translational modification profiling. We present fast and robust SP3- and STRAP-based protocols for the broad-specificity proteases subtilisin, proteinase K, and thermolysin. All three enzymes are remarkably fast, producing near-complete digests in 1-5 min, and cost 200-1000× less than proteomics-grade trypsin. Using FragPipe resolved a major challenge by drastically reducing the duration of the required "unspecific" searches. In-depth analyses of proteinase K, subtilisin, and thermolysin Jurkat digests identified 7374, 8178, and 8753 unique proteins with average sequence coverages of 21, 29, and 37%, including 10,000s of amino acids not reported in PeptideAtlas' >2400 experiments. While we could not identify distinct cleavage patterns, machine learning could distinguish true protease products from random cleavages, potentially enabling the prediction of cleavage products. Finally, proteinase K, subtilisin, and thermolysin enabled label-free quantitation of 3111, 3659, and 4196 unique Jurkat proteins, which in our hands is comparable to trypsin. Our data demonstrate that broad-specificity proteases enable quantitative proteomics of uncharted areas of the proteome. Their fast kinetics may allow "on-the-fly" digestion of samples in the future.