Biology subjects

Elsborg, J. D.

Publications and source records attributed to Elsborg, J. D..

4 recordsLinked to original sources

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↗

bioRxiv2025

Deciphering cytokine-driven ADP-ribosylation signaling networks via Af1521-based mass spectrometry analysis of labile Glu/Asp-linkages

ADP-ribosylation (ADPr) is a regulatory post-translational modification targeting nine amino acid residues. Glutamate- and aspartate-linked ADPr (Glu/Asp-ADPr) is chemically unstable during sample preparation for conventional mass spectrometry (MS)-based proteomics workflows, limiting its detection. Here, we systematically assessed the stability of ADPr linkages using synthetic peptides and confirmed that ester-linked Glu/Asp-ADPr is lost under alkaline conditions, elevated temperatures, or through hydrolysis by wildtype Af1521. We established an acidic enrichment workflow encompassing an Af1521 mutant, robustly preserving Glu/Asp-ADPr enabling their site-specific and systems-wide MS analysis. Applying this strategy to cytokine-stimulated A549 and HeLa cells, we identified >600 Glu/Asp-ADPr and >200 Cys-ADPr sites. Our analysis uncovered that Glu/Asp-ADPr marks distinct cytoplasmic protein networks enriched in immune functions, contrasting with Ser-ADPr typically observed on nuclear and chromatin-associated proteins. Quantitative profiling revealed reproducible ADPr patterns specific to cell type and treatment. Notably, PARP10 promoted Glu/Asp modification of ubiquitin, highlighting crosstalk between ADPr and ubiquitin signaling. Across interferon treatments, we identified a conserved network of antiviral PARPs and associated cofactors extensively modified on Glu/Asp residues, emphasizing residue-specific ADPr as a regulator of innate immune signaling. Together, our work establishes an MS-based proteomics workflow for identification of Glu/Asp-ADPr, provides a resource of site-specific modification events, and reveals residue-specific ADPr dynamics in immune signaling.

cell biology↗

bioRxiv2022

A quantitative and site-specific atlas of the in vivo citrullinome reveals widespread existence of citrullination

Citrullination is the conversion of peptidyl-arginine into the non-coded amino acid citrulline. Despite its importance in physiology and disease, global identification of citrullinated proteins and precise modification sites has remained challenging. Here, we employed quantitative mass spectrometry-based proteomics to generate a comprehensive atlas of citrullination sites in a physiologically relevant cell type. Collectively, we identified 14.056 citrullination sites within 4.008 proteins and quantified their regulation upon inhibition of the citrullinating enzyme PADI4. Using this rich dataset, we uncover general mechanistic and cell biological principles of citrullination function, while providing site-specific and quantitative information on thousands of PAD4 substrates within cells. Our findings include signature histone marks and numerous modifications on transcriptional regulators and chromatin-related signaling effectors. Additionally, we identify precise citrullination sites on an extensive list of known autoantigens. Collectively, we describe systems attributes of the human citrullinome and provide a resource framework for understanding citrullinaiton at the mechanistic level.

molecular biology↗

bioRxiv2022

DAXX adds a de novo H3.3K9me3 deposition pathway to the histone chaperone network

A multitude of histone chaperones are required to protect histones after their biosynthesis until DNA deposition. They cooperate through the formation of co-chaperone complexes, but the crosstalk between nucleosome assembly pathways remains enigmatic. Using explorative interactomics approaches, we characterize the organization of the histone H3-H4 chaperones network and define the interplay between histone chaperone systems. We identify and validate several novel histone dependent complexes and predict the structure of the ASF1 and SPT2 co-chaperone complex, expanding the role of ASF1 in histone dynamics. We show that DAXX acts separately from the rest of the network, recruiting heterochromatin factors and promoting lysine 9 tri-methylation of new histone H3.3 prior to deposition onto DNA. With its functionality, DAXX provides a molecular mechanism for de novo heterochromatin assembly. Collectively, our findings provide a new framework for understanding how cells orchestrate histone supply and comply with chromatin dynamics throughout the cell cycle.

molecular biology↗