Comparative Analysis of Lysine-Specific Peptidases for Optimizing Proteomics Workflows

This study presents a comparative analysis of three LysC endopeptidase homologues from Achromobacter lyticus (A. lyticus),Pseudomonas aeruginosa and Lysobacter enzymogenes for mass spectrometry-based proteomics. Utilizing a protein aggregation capture workflow with HeLa cell lysates, we assessed the enzymes’ cleavage specificity, digestion efficiency, and performance across various experimental conditions. Results showed that while all three LysC homologues exhibited high cleavage specificity at lysine residues, A. lyticus LysC outperformed the two others with superior peptide identification, digestion efficiency, and protein coverage, especially at shorter digestion times. Our experiments using a combination ofA. lyticusLysC and trypsin demonstrated the importance of employing LysC for significantly minimizing missed cleavage rates in tryptic digests, especially with regard to lysine-containing peptides. This study underscores A. lyticus LysC’s potential as an optimal choice for enhancing mass spectrometry-based proteomics.

pubs.acs.org

OVA-PDM - Personalising clinical decisions in ovarian cancer through patient-derived in vitro models

YouTube video by European Partnership for Personalised Medicine

youtu.be

Higher-Throughput Proteome Profiling Enabled by Parallelized Pre-Accumulation and Optimized Ion Processing in the Orbitrap Astral Zoom Mass Spectrometer

High-throughput proteomics is critical for understanding biological processes, enabling large-scale studies such as biomarker discovery and systems biology. However, current mass spectrometry technolo...

biorxiv.org

Enhancing Tandem MS Sensitivity and Peptide Identification via Ion Preaccumulation in an Orbitrap Mass Spectrometer

High-throughput mass spectrometry-based proteomics has gained increasing interest for both academic and industrial applications. As implementation of faster gradients has facilitated higher sample thr...

pubs.acs.org

Proteomics of colorectal tumors identifies the role of CAVIN1 in tumor relapse | Molecular Systems Biology

imageimageProteomics profiling of a pre-metastatic colorectal cancer cohort identifies four proteomics-based subtypes and highlights the role of CAVIN1 in relapse in EMT-like tumors. Mass-spectrometr...

embopress.org

Quantitative proteomics and phosphoproteomics analysis of patient-derived ovarian cancer stem cells

High-grade serous ovarian carcinoma (HGSOC) is the deadliest gynecologic cancer. Key to the progression and ultimate lethality of this subtype is the intra-tumoral heterogeneity (ITH), which is define...

mcponline.org

Global analysis of protein turnover dynamics in single cells

The SC-pSILAC method enables single-cell measurement of both protein abundance and turnover, providing notable advances in the depth and versatility of proteomic technologies.

eur02.safelinks.protection.outlook.com

Formaldehyde Fixation Helps Preserve the Proteome State during Single-Cell Proteomics Sample Processing and Analysis

Mass spectrometry-based single-cell proteomics (SCP) is gaining momentum but remains limited to a few laboratories due to the high costs and specialized expertise required. The ability to send samples to specialized core facilities would benefit nonspecialist laboratories and popularize SCP for biological applications. However, no methods have been tested in SCP to “freeze” the proteome state while maintaining cell integrity for transfer between laboratories or prolonged sorting using fluorescence-activated cell sorting (FACS). This study evaluates whether short-term formaldehyde (FA) fixation can maintain the cell states. We demonstrate that short-term FA fixation does not substantially affect protein recovery, even without heating and strong detergents, and maintains analytical depth compared with classical workflows. Fixation also preserves drug-induced specific perturbations of the protein abundance during cell sorting and sample preparation for SCP analysis. Our findings suggest that FA fixation can facilitate SCP by enabling sample shipping and prolonged sorting, potentially democratizing access to SCP technology and expanding its application in biological research, thereby accelerating discoveries in cell biology and personalized medicine.

pubs.acs.org

Enhanced sensitivity and scalability with a Chip-Tip workflow enables deep single-cell proteomics - Nature Methods

Chip-Tip is a label-free quantification-based single-cell proteomics workflow for deep single-cell proteomics, which identifies over 5,000 proteins and 40,000 peptides in single HeLa cells.

nature.com