Biology subjects

Lancaster, N. M.

Publications and source records attributed to Lancaster, N. M..

5 recordsLinked to original sources

bioRxiv2026

Atlas of lysine acetylation in the mouse

Lysine acetylation has widespread ramifications from genetic regulatory effects to modulation of enzymatic function. With improved acetyl-lysine enrichment technologies and advances in mass spectrometer speed and sensitivity, we present a comprehensive atlas of the mouse acetylome comprising 17,952 unique lysine acetylation sites across 4,340 proteins and 15 tissues. This resource, which nearly doubles the known mouse acetyl-lysine catalog, shows at least 14% of the acetylome is shared across tissues. We focus our investigation on several acetylated proteins, including ribosomal acetylation and its potential to extend ribosomal half-life in the liver and pancreas. Additionally, we identify a novel acetylation event in the active site of carnitine O-acetyltransferase (Crat) that also mirrors tissue-specific Crat activity. By integrating these data with human pathogenic variants, we identify acetyl-lysine residues on cardiac troponin and homogentisate dioxygenase that likely mimic disease-causing mutations. This resource provides a foundational framework for investigating protein acetylation in metabolic health and disease.

biochemistry↗

bioRxiv2025

Regarding Emitter Positioning for Nanoflow Electrospray Ionization

Nanoflow electrospray ionization is commonly used for proteomics due to its high sensitivity. Signal intensity, however, is dependent on optimal emitter positioning relative to the mass spectrometer inlet. Here, we characterize the effect of varied emitter positions on peptide signal intensity in all three dimensions using emitters and flows consistent with standard proteomic analyses. We observe improved signal robustness to x/y variations at increasing z distances and demonstrate that positioning within 1 to 2 mm of the optimal location will maintain consistent signal. Signal intensity behavior is consistent across the m/z range, suggesting a certain level of analyte-independence for proteomics analyses. These results provide insight for proteomics researchers using nanoflow LC-MS/MS. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/695161v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1a976ccorg.highwire.dtl.DTLVardef@b6627forg.highwire.dtl.DTLVardef@1fca93eorg.highwire.dtl.DTLVardef@cb61f6_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

A Technical Evaluation of Plasma Proteomics Technologies

Plasma proteomic technologies are rapidly evolving and of critical importance to the field of biomedical research. Here we report a technical evaluation of six notable plasma proteomic technologies - unenriched (Neat), Acid depletion, PreOmics ENRICHplus, Mag-Net, Seer Proteograph XT, Olink Explore HT. The methods were compared on proteomic depth, reproducibility, linearity, tolerance to lipid interference, and limit of detection/quantification. In total we performed 618 LC-MS/MS experiments and 93 Olink Explore HT assays. The Seer method achieved the greatest proteomic depth ([~]4,500), while Olink detected [~]2,600 proteins. Other MS-based methods ranged from [~]500-2,200. Neat, Mag-Net, Seer, and Olink had strong reproducibility, while PreOmics and Acid showed higher variability. All MS methods showed good linearity with spiked-in C-Reactive Protein (CRP); CRP was surprisingly not in the Olink assay. None of the methods were affected by lipid interference. Seer had more than double the number of quantifiable proteins (4,800) for both LOD and LOQ than the next best method. Olink was comparable to Neat and Mag-Net for LOD, but worse for LOQ. Finally, we tested the applicability of these methods for detecting differences between healthy and cancer groups in a non-small cell lung cancer (NSCLC) cohort.

systems biology↗

bioRxiv2024

SPE-CZE-MS quantifies zeptomole concentrations ofphosphorylated peptides

Capillary zone electrophoresis (CZE) is gaining attention in the field of single-cell proteomics for its ultra-low-flow and high-resolution separation abilities. Even more sample-limited yet rich in biological information are phosphoproteomics experiments, as the phosphoproteome composes only a fraction of the whole cellular proteome. Rapid analysis, high sensitivity, and maximization of sample utilization are paramount for single-cell analysis. Some challenges of coupling CZE analysis with mass spectrometry analysis (MS) of complex mixtures include 1. sensitivity due to volume loading limitations of CZE and 2. incompatibility of MS duty cycles with electrophoretic timescales. Here, we address these two challenges as applied to single-cell equivalent phosphoproteomics experiments by interfacing a microchip-based CZE device integrated with a solid-phase-extraction (SPE) bed with the Orbitrap Astral mass spectrometer. Using 225 phosphorylated peptide standards and phosphorylated peptide-enriched mouse brain tissue, we investigate microchip-based SPE-CZE functionality, quantitative performance, and complementarity to nano-LC-MS (nLC-MS) analysis. We highlight unique SPE-CZE separation mechanisms that can empower fit-for-purpose applications in single-cell-equivalent phosphoproteomics.

systems biology↗

bioRxiv2023

One-Hour Phosphoproteome Analysis with the Orbitrap Astral Mass Spectrometer

Owing to its roles in cellular signal transduction, protein phosphorylation plays critical roles in myriad cell processes. That said, detecting and quantifying protein phosphorylation has remained a challenge. We describe the use of a novel mass spectrometer (Orbitrap Astral) coupled with data-independent acquisition (DIA) to achieve rapid and deep analysis of human and mouse phosphoproteomes. With this method we map approximately 30,000 unique human phosphorylation sites within a half-hour of data collection. The technology was benchmarked to other state-of-the-art MS platforms using both synthetic peptide standards and with EGF-stimulated HeLa cells. We applied this approach to generate a phosphoproteome multi-tissue atlas of the mouse. Altogether, we detected 81,120 unique phosphorylation sites within 12 hours of measurement. With this unique dataset, we examine the sequence, structural, and kinase specificity context of protein phosphorylation. Finally, we highlight the discovery potential of this resource with multiple examples of novel phosphorylation events relevant to mitochondrial and brain biology.

systems biology↗