Biology subjects

Hernandez-Rollan, C.

Publications and source records attributed to Hernandez-Rollan, C..

4 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

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↗

bioRxiv2020

LyGo: A platform for rapid screening of lytic polysaccharide monooxygenase production

Environmentally friendly sources of energy and chemicals are essential constituents of a sustainable society. An important step towards this goal is the utilization of non-edible biomass as supply of building blocks for future biorefineries. Lytic polysaccharide monooxygenases (LPMOs) are enzymes that play a critical role in breaking the chemical bonds in the most abundant polymers found in recalcitrant biomass, such as cellulose and chitin. Predicting optimal strategies for producing LPMOs is often non-trivial, and methods allowing for screening several strategies simultaneously are therefore needed. Here, we present a standardized platform for cloning LPMOs. The platform allows users to combine gene fragments with different expression vectors in a simple 15-minute reaction, thus enabling rapid exploration of several gene contexts, hosts and expression strategies in parallel. The open-source LyGo platform is accompanied by easy-to-follow online protocols for both cloning and expression. As a demonstration, we utilize the LyGo platform to explore different strategies for expressing several different LPMOs in Escherichia coli, Bacillus subtilis, and Komagataella phaffii.

bioengineering↗