Biology subjects

Batth, T. S.

Publications and source records attributed to Batth, T. S..

9 recordsLinked to original sources

bioRxiv2026

High-fidelity bispecific protease KRysin for deeper plasma proteomics and phosphoproteomics

Bottom-up proteomics relies on efficient and reproducible proteolytic digestion. Yet trypsin, the most widely used protease, frequently misses cleavages, particularly near prolines, acidic and phosphorylated residues. Incomplete digestion inflates search space, dilutes peptide signal, and limits depth and reproducibility, especially in challenging matrices such as plasma and phosphoproteomics. Here we present KRysin, a recombinant bispecific protease that cleaves C-terminal to lysine and arginine with very high specificity and efficiency, generating tryptic peptides compatible with standard proteomics workflows. In HeLa digests, ~99% of KRysin-generated peptides ended in lysine or arginine, including at sites followed by proline, and only 0.2% of peptides contained an internal (missed-cleaved) arginine and 6% an internal lysine, compared with 9% and 25%, respectively, for trypsin. Using narrow-window DIA at 60 samples-per-day, KRysin quantified close to 50,000 class I phosphosites, >10,000 more than trypsin-based workflows, and identified up to 40% more plasma protein groups, uniquely revealing receptors, cytokines, and growth factors. KRysin therefore provides a direct alternative to trypsin wherever digestion completeness, speed and PTM coverage are limiting.

biochemistry↗

bioRxiv2026

Mapping the human sperm proteome across compartments, donors and individual cells

The human spermatozoon is among the most specialized cells in the body, yet where its proteins reside, where they come from and how they vary between and within men remain largely unresolved. Here we present the deepest proteomic atlas of human spermatozoa to date, identifying 10,828 proteins across six donors. Abundances correlate well between individuals and are anchored by a conserved core of mitochondrial energy-metabolism proteins, while a smaller fraction varies between donations from the same man. By mechanically separating heads from tails, we assigned thousands of previously unlocalized proteins to a compartment, and by comparing seminal fluid from vasectomized and non-vasectomized men we distinguished sperm- and epididymis-derived proteins from accessory-gland secretions. Using a single-cell analysis workflow we further show that the proteomes of individual spermatozoa can be measured, recovering up to ~1,000 protein groups per cell. In a cohort with total fertilization failure, we find recurrent mitochondrial rather than sperm-egg fusion dysregulation. Together or data provides novel insights into the dynamics of the sperm proteome and male infertility.

cell biology↗

bioRxiv2026

Near-Zero Missed Cleavages with a High-Fidelity Recombinant Arg-C Zero for Mass Spectrometry-Based Proteomics

Proteolytic digestion remains a critical step in bottom-up proteomics workflows, with enzyme specificity and efficiency directly impacting peptide identification and protein sequence coverage. Here, we present the comprehensive characterization of Arg-C Zero, a recombinant arginyl endopeptidase derived from Porphyromonas gingivalis that exhibits exceptional fidelity in cleaving specifically at the C-terminus of arginine residues. Unlike conventional serine proteases such as Trypsin, Arg-C Zero utilizes a histidine-cysteine catalytic dyad mechanism, achieving near-zero missed cleavage rates (>99% efficiency) under standard proteomics conditions. Through systematic evaluation using HeLa protein extracts, we demonstrate that Arg-C Zero maintains consistent performance across varying digestion times. The enzyme shows robust activity across a broad pH range and tolerates up to 4M urea, making it ideally suitable for a diverse range of proteomics sample preparation workflows. While Trypsin/LysC combinations remain superior for comprehensive proteome coverage, Arg-C Zero offers unique advantages for applications requiring high specificity and reproducible arginine-specific cleavage patterns, particularly for analysis of post-translational modifications (PTMs). Here, we demonstrate how Arg-C Zero aids comprehensive mapping of histone PTMs, and when used in low-pH workflows help preserve labile ADP-ribosylation sites, expanding the analytical capabilities of mass spectrometry for characterizing these challenging modifications. The enzymes resistance to proline-adjacent cleavage sites and compatibility with standard mass spectrometry buffers position it as a valuable addition to the proteomics enzyme toolkit.

biochemistry↗

bioRxiv2024

Comparative Analysis of Lysine-Specific Peptidases for Optimizing Proteomics Workflows

This study presents a comparative analysis of three LysC homologues from Achromobacter lyticus, Pseudomonas aeruginosa, and Lysobacter enzymogenes for mass spectrometry-based proteomics. Utilizing a protein aggregation capture (PAC) workflow with HeLa cell lysate, we assessed the enzymes cleavage specificity, digestion efficiency, and performance across various experimental conditions. Results showed that while all three homologues exhbihited high cleavage specificity at lysine residues, A. lyticus LysC outperformed the two others with its superior peptide identification, digestion efficiency, and protein coverage, especially at short digestion times. Combination of A. lyticus LysC and trypsin demonstrated that importance of LysC for signifancanlty minimizing missed cleavage rates in tryptic digests. This study underscores A. lyticus LysCs potential as an optimal choice for enhancing mass spectrometry-based proteomics.

biochemistry↗

bioRxiv2024

STREAMLINED PROTEOME-WIDE IDENTIFICATION OF DRUG TARGETS INDICATES ORGAN-SPECIFIC ENGAGEMENT

Proteins are the primary targets of almost all small molecule drugs. However, even the most selectively designed drugs can potentially target several unknown proteins. Identification of potential drug targets can facilitate design of new drugs and repurposing of existing ones. Current state-of-the-art proteomics methodologies enable screening of thousands of proteins against a limited number of drug molecules. Here we report the development of a label-free quantitative proteomics approach that enables proteome-wide screening of small organic molecules in a scalable, reproducible, and rapid manner by streamlining the proteome integral solubility alteration (PISA) assay. We used rat organs ex-vivo to determine organ specific targets of medical drugs and enzyme inhibitors to identify novel drug targets for common drugs such as Ibuprofen. Finally, global drug profiling revealed overarching trends of how small molecules affect the proteome through either direct or indirect protein interactions.

biochemistry↗

bioRxiv2022

A seven transmembrane-integral methyltransferase catalysing N-terminal histidine methylation of lytic polysaccharide monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes that help break down lignocellulose, making them highly attractive for improving biomass utilization in biotechnological purposes. The catalytically essential N-terminal histidine (His1) of LPMOs is post-translationally modified by methylation in filamentous fungi to protect them from auto-oxidative inactivation, however, the responsible methyltransferase enzyme is unknown. Using mass-spectrometry-based quantitative proteomics in combination with systematic CRISPR/Cas9 knockout screening in Aspergillus nidulans, we identified the N-terminal histidine methyltransferase (NHMT) encoded by the gene AN4663. Targeted proteomics confirmed that NHMT was solely responsible for His1 methylation of LPMOs. NHMT is predicted to encode a unique seven-transmembrane segment anchoring a soluble methyltransferase domain. Co-localization studies showed endoplasmic reticulum residence of NHMT and co-expression in the industrial production yeast Komagataella phaffii with LPMOs resulted in His1 methylation of the LPMOs. This demonstrates the biotechnological potential of recombinant production of proteins and peptides harbouring this unique post-translational modification.

molecular biology↗

bioRxiv2019

Proteome and phosphoproteome changes associated with prognosis in acute myeloid leukemia

Acute myeloid leukemia (AML) is a hematological cancer that mainly affects the elderly. Although complete remission (CR) is achieved for majority of the patients after induction and consolidation therapies, nearly two-thirds relapse within a short interval. Understanding biological factors that determine relapse has therefore become of major clinical interest in AML. We utilized liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify protein changes and protein phosphorylation events associated with AML relapse in primary cells from 41 AML patients at time of diagnosis. Patients were defined as relapse-free if they had not relapsed within a 5-year clinical follow-up after AML diagnosis. Relapse was associated with increased expression of RNA processing proteins and decreased expression of V-ATPase proteins. We also observed an increase in phosphorylation events catalyzed by cyclin-dependent kinases (CDKs) and casein kinase 2 (CSK2). The biological relevance of the proteome findings was supported by cell proliferation assays using inhibitors of V-ATPase (bafilomycin), CSK2 (CX-4945), CDK4/6 (abemaciclib) and CDK2/7/9 (SNS-032). While bafilomycin preferentially inhibited the cells from relapse patients, the kinase inhibitors were less efficient in these cells. This suggests that therapy against the upregulated kinases also could target the factors inducing their upregulation rather than their activity. In conclusion, our study presents markers that could help predict AML relapse and direct therapeutic strategies.

cancer biology↗

bioRxiv2018

Protein aggregation capture on microparticles enables multi-purpose proteomics sample preparation

Universal proteomics sample preparation is challenging due to the high heterogeneity of biological samples. Here we describe a novel mechanism that exploits the inherent instability of denatured proteins for non-specific immobilization on microparticles by protein aggregation capture. To demonstrate the general applicability of this mechanism, we analyzed phosphoproteomes, tissue proteomes, and interaction proteomes as well as dilute secretomes. The findings presents a practical, sensitive and cost-effective proteomics sample preparation method.

biochemistry↗

bioRxiv2018

Limits for resolving tandem mass tag reporter ions with identical integer mass using phase constrained spectrum deconvolution

A popular method for peptide quantification relies on isobaric labeling such as tandem mass tags (TMT) which enables multiplexed proteome analyses. Quantification is achieved by reporter ions generated by fragmentation in a tandem mass spectrometer. However, with higher degrees of multiplexing, the smaller mass differences between the reporter ions increase the mass resolving power requirements. This contrasts with faster peptide sequencing capabilities enabled by lowered mass resolution on Orbitrap instruments. It is therefore important to determine the mass resolution limits for highly multiplexed quantification when maximizing proteome depth. Here we defined the lower boundaries for resolving TMT reporter ions with 0.0063 Da mass differences using an ultra-high-field Orbitrap mass spectrometer. We found the optimal method depends on the relative ratio between closely spaced reporter ions and that 64 ms transient acquisition time provided sufficient resolving power for separating TMT reporter ions with absolute ratio changes up to 16-fold. Furthermore, a 32 ms transient processed with phase-constrained spectrum deconvolution provides >50% more identifications with >99% quantified, but with a slight loss in quantification precision and accuracy. These findings should guide decisions on what Orbitrap resolution settings to use in future proteomics experiments relying on TMT reporter ion quantification with identical integer masses.

biochemistry↗