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Shotgun proteomics uses liquid chromatography–tandem mass spectrometry to identify proteins in complex biological samples. We describe an algorithm, called Percolator, for improving the rate of confident peptide identifications from a collection of tandem mass spectra. Percolator uses semi-supervised machine learning to discriminate between correct and decoy spectrum identifications, correctly assigning peptides to 17% more spectra from a tryptic Saccharomyces cerevisiae dataset, and up to 77% more spectra from non-tryptic digests, relative to a fully supervised approach.

Mediators involved in the generation of symptoms in patients with irritable bowel syndrome (IBS) are poorly understood. Here we show that colonic biopsy samples from IBS patients release increased levels of proteolytic activity (arginine cleavage) compared to asymptomatic controls. This was dependent on the activation of NF-kappaB. In addition, increased proteolytic activity was measured in vivo, in colonic washes from IBS compared with control patients. Trypsin and tryptase expression and release were increased in colonic biopsies from IBS patients compared with control subjects. Biopsies from IBS patients (but not controls) released mediators that sensitized murine sensory neurons in culture. Sensitization was prevented by a serine protease inhibitor and was absent in neurons lacking functional protease-activated receptor-2 (PAR2). Supernatants from colonic biopsies of IBS patients, but not controls, also caused somatic and visceral hyperalgesia and allodynia in mice, when administered into the colon. These pronociceptive effects were inhibited by serine protease inhibitors and a PAR2 antagonist and were absent in PAR2-deficient mice. Our study establishes that proteases are released in IBS and that they can directly stimulate sensory neurons and generate hypersensitivity symptoms through the activation of PAR2.

Background and Objectives: Acute pancreatitis (AP) has a wide range of clinical severity, and early prediction of disease progression is still challenging. Trypsinogen-activating peptide (TAP) and trypsin-2 serve as direct biomarkers for intrapancreatic proteolytic activation and may provide earlier pathophysiological information compared with traditional markers. Materials and Methods: In this retrospective cohort analysis involving 54 AP patients, we evaluated 24 h serum and urinary TAP and trypsin-2 concentrations by Bayesian correlation, mediation analysis, unsupervised K-means clustering, and supervised machine learning (Elastic Net and Random Forest). The analyses investigated the relationships of biomarkers with inflammation (CRP), enzymatic activities (amylase, lipase), and clinical factors, as well as inflammation severity (CRP levels). Results: Bayesian correlations indicated moderate evidence for a relationship between serum TAP and CRP (BF10 = 8.42), as well as strong evidence linking age to serum TAP (BF10 = 12.75). Serum trypsin-2 showed no correlation with CRP, while urinary trypsin-2 had a correlation with amylase (BF10 = 6.89). Mediation analysis indicated that TAP and trypsin-2 accounted for 42–44% of the impact of CRP on pancreatic enzyme elevation. Clustering revealed three phenotypic subgroups (“Mild Activation”, “Moderate System”, and “Severe Pancreatic-Renal”), the latter showing the highest levels of CRP and biomarkers. Machine learning models highlighted urinary trypsin-2 and age as the most significant predictors of inflammation, with Random Forest achieving the highest performance (R2 = 0.53). Conclusions: Early urinary trypsin-2 outperforms serum markers as a predictor of systemic inflammatory intensity, indicating total proteolytic impairment and renal clearance. This integrative analysis reveals unique biological phenotypes and highlights the potential of these biomarkers for early assessment of the inflammatory burden. Their role in predicting clinical disease progression requires prospective validation. Integrative biomarker analysis reveals unique biological phenotypes and improves assessment of inflammatory burden in PA. Larger cohorts are required for prospective validation to incorporate these biomarkers into precision-based diagnostic frameworks.

A dual-signal assay is described for the determination of trypsin based on the use of gold nanoparticles (AuNPs) that aggregate in the presence of gold nanoclusters (AuNCs) due to electrostatic interaction. This is accompanied by a color change from red to blue. However, if hemoglobin (Hb) is present in the solution, it will attach to the surface of AuNPs, thus preventing aggregation. The Hb-coated AuNPs quench the fluorescence of AuNCs. Trypsin can hydrolyze Hb and destroy the protective coating of Hb on the AuNPs. As a result, AuNP aggregation will occur after the addition of AuNCs, and the blue fluorescence of the AuNCs with 365 nm excitation and 455 nm maximum emission peak is recovered. Thus, trypsin can be determined by measurement of fluorescence emission intensity. Additionally, trypsin can be determined by the maximum absorption peak wavelength between 530 nm and 610 nm. Fluorescence increases linearly in the 10–2500 ng⋅mL −1 concentration range, and absorbance in the 20–2000 ng·mL −1 concentration range. The limits of detection are 4.6 ng·mL −1 (fluorometry) and 8.4 ng·mL −1 (colorimetry), respectively. The assay is sensitive and selective, and can be applied to the determination of trypsin in serum. Graphical abstract Schematic presentation of a fluorometric and colorimetric method for determination of trypsin. The presence of hemoglobin (Hb) protects AuNPs from agglomeration after adding AuNCs and the fluorescence of AuNCs is quenched. With trypsin present, trypsin destroys the coating of AuNPs by Hb. AuNPs aggregate again and the fluorescence recovers after the addition of AuNCs.

Protein homeostasis is an emerging component of schizophrenia (SZ) pathophysiology. Proteomic alterations in SZ are well-documented and changes in transcript expression are frequently not associated with changes in protein expression in SZ brain. The underlying mechanism driving these changes remains unknown, though altered expression of ubiquitin proteasome system (UPS) components have implicated protein degradation. Previous studies have been limited to protein and transcript expression, however, and do not directly test the function of the proteasome. To address this gap in knowledge, we measured enzymatic activity associated with the proteasome (chymotrypsin-, trypsin-, and caspase-like) in the superior temporal gyrus (STG) of 25 SZ and 25 comparison subjects using flourogenic substrates. As localization regulates which cellular processes the proteasome contributes to, we measured proteasome activity and subunit expression in fractions enriched for nucleus, cytosolic, and membrane compartments. SZ subjects had decreased trypsin-like activity in total homogenate. This finding was specific to the nucleus-enriched fraction and was not associated with changes in proteasome subunit expression. Interestingly, both chymotrypsin-like activity and protein expression of 19S RP subunits, which facilitate ubiquitin-dependent degradation, were decreased in the cytosol-enriched fraction of SZ subjects. Intracellular compartment-specific proteasome dysfunction implicates dysregulation of protein expression both through altered ubiquitin-dependent degradation of cytosolic proteins and regulation of protein synthesis due to degradation of transcription factors and transcription machinery in the nucleus. Together, these findings implicate proteasome dysfunction in SZ, which likely has a broad impact on the proteomic landscape and cellular function in the pathophysiology of this illness.

An electrostatically controlled fluorometric assay is described that is based on the use of silver/copper bimetallic nanoclusters. The nanoclusters were coated with polyethyleneimine (PEI-Ag/CuNCs). At pH 7.4, these particles are positively charged. Their blue fluorescence (with excitation/emission peaks at 341/464 nm) depends on local pH values and temperature. If graphene oxide (which is negatively charged at pH 7.4) is introduced, the fluorescence of the PEI-Ag/CuNCs is quenched. Based on various electrostatic interactions, three kinds of biomacromolecules were detected by fluorometry. These include (negatively charged) heparin, (positively charged) protamine, and (virtually uncharged) trypsin. Heparin is detected by using GO/PEI-Ag/CuNCs, protamine by using GO/heparin/PEI-Ag/CuNCs, and trypsin by using GO/protamine/heparin/PEI-Ag/CuNC. The detection limits and linear ranges are 4.8 nM and 10–450 nM for heparin, 0.09 μg·mL −1 and 0.25–5 μg·mL −1 for protamine, and 0.03 μg·mL −1 and 0.05–1 μg·mL −1 for trypsin. Zeta potentials of the various substances in the system were determined to elucidate the detection mechanism. Comceivably, the method provides a widely applicable approach for electrostatically controlled biomolecular assays. Graphical abstract Schematic presentation of electrostatically controlled fluorometric assay for the detection of heparin, protamine, and trypsin based on the silver/copper bimetallic nanoclusters modified with polyethyleneimine and graphene oxide.

Stable copper nanoclusters (CuNCs) were prepared by utilizing D-penicillamine as both the stabilizer and reductant. The emission of the CuNCs (with excitation/emission peaks at 390/645 nm) is largely stabilized by coating with poly(sodium-p-styrenesulfonate) (PSS). Cytochrome c (Cyt c) quenches the fluorescence of the PSS-coated CuNCs, and this effect was exploited to design a quenchometric fluorometric assay for Cyt c. If trypsin is added to the loaded CuNCs, it will hydrolyze Cyt c to form peptide fragments, and fluorescence is gradually restored. A highly sensitive and fluorometric turn-off-on assay was constructed for sequential detection of Cyt c and trypsin. The linear ranges for Cyt c and trypsin are from 8.0 nM to 680 nM, and from 0.1 to 6.0 μg mL −1 , and the lower detection limits are 0.83 nM and 20 ng mL −1 for Cyt c and trypsin, respectively. Graphical abstract Schematic illustration of the fluorometric assay for trypsin based on the electron transfer between poly(p-styrenesulfonate)-protected copper nanoclusters (PSS-CuNCs) and cytochrome c (Cyt c).

Trypsin and chymotrypsin are both serine proteases with high sequence and structural similarities, but with different substrate specificity. Previous experiments have demonstrated the critical role of the two loops outside the binding pocket in controlling the specificity of the two enzymes. To understand the mechanism of such a control of specificity by distant loops, we have used the Gaussian network model to study the dynamic properties of trypsin and chymotrypsin and the roles played by the two loops. A clustering method was introduced to analyze the correlated motions of residues. We have found that trypsin and chymotrypsin have distinct dynamic signatures in the two loop regions, which are in turn highly correlated with motions of certain residues in the binding pockets. Interestingly, replacing the two loops of trypsin with those of chymotrypsin changes the motion style of trypsin to chymotrypsin-like, whereas the same experimental replacement was shown necessary to make trypsin have chymotrypsin's enzyme specificity and activity. These results suggest that the cooperative motions of the two loops and the substrate-binding sites contribute to the activity and substrate specificity of trypsin and chymotrypsin.

Amylase/trypsin inhibitors (ATIs) are major wheat allergens and they are also implicated in causing non-celiac gluten sensitivity and worsening other inflammatory conditions. With only few studies on ATI contents in different Triticum species available so far, we developed a targeted liquid chromatography-tandem mass spectrometry (LC–MS/MS) method based on stable isotope dilution assays to quantitate the 13 most important ATIs in a well-defined sample set of eight cultivars of common wheat and durum wheat (modern species), as well as spelt, emmer and einkorn (ancient species) grown at three locations in Germany, respectively. Only few ATIs with low contents were detected in einkorn. In contrast, spelt had the highest total ATI contents. Emmer and common wheat had similar total ATI contents, with durum wheat having lower contents than common wheat. Due to the lack of correlation, it was not possible to estimate ATI contents based on crude protein contents. The wheat species had a higher influence on ATI contents than the growing location and the heritability of this trait was high. Despite comparatively low intra-species variability, some cultivars were identified that may be promising candidates for breeding for naturally low ATI contents.

Gold nanocluster@carbon nitride quantum dot nanocomposites protected by bovine serum albumin (BSA-AuNC@CNQDs) were designed as a ratiometric fluorescence nanosensor for ultra-sensitive detection of trypsin inhibitor (TI). CNQDs were prepared via thermal treatment of carbon nitride powder. BSA-CNQDs acted as templates to synthesize BSA-AuNC@CNQDs with dual-emission peaks at 450 and 650 nm. Trypsin can catalyze the hydrolysis of BSA and decompose BSA-AuNC@CNQDs resulting in fluorescence quenching. The fluorescence quenching at 650 nm was prevented by the addition of TI to inhibit the activity of trypsin. The nanosensor-trypsin system showed a satisfactory ability toward TI detection. The ratiometric responses (the ratio of intensity at 650 to 450 nm, I650/I450) had an excellent linearity (R2 = 0.981) with logarithmic values of TI concentrations in the broad range of 1–10,000 ng/mL. The limit of detection (LOD, 0.089 ng/mL) indicates ultra-sensitive detection of TI can be achieved. Additionally, TI in soybean flour was detected by the proposed ratiometric method with satisfactory recoveries (98.15–105.52%) and less than 6% of coefficient of variation. This study reveals that BSA-AuNC@CNQDs have potential applications in detection of TI in real samples.