In attempting to observe the behaviour of a protein of interest in vivo, akin to other observer effects, current techniques require modifications or interventions that inherently alter the protein or ...

Poor repeatability of peak areas is a problem frequently encountered in peptide analysis with nanoLiquid Chromatography coupled on-line with Mass Spectrometry (nanoLC−MS). As a result, quantitative an...

In this work, we describe the construction and application of a repurposed 3D-printer as a fraction collector. We utilize a nano-LC to ensure minimal volumes and surfaces although any LC can be coupled. The setup operates as a high-pH fractionation system capable of effectively working with nanogram scales of lysate digests. The 2D RP–RP system demonstrated superior proteome coverage over single-shot data-dependent acquisition (DDA) analysis using only 5 ng of human cell lysate digest with performance increasing with increasing amounts of material. We found that the fractionation system allowed over 60% signal recovery at the peptide level and, more importantly, we observed improved protein level intensity coverage, which indicates the complexity reduction afforded by the system outweighs the sample losses endured. The application of data-independent acquisition (DIA) and wide window acquisition (WWA) to fractionated samples allowed nearly 8000 proteins to be identified from 50 ng of the material. The utility of the 2D system was further investigated for phosphoproteomics (>21 000 phosphosites from 50 μg starting material) and pull-down type experiments and showed substantial improvements over single-shot experiments. We show that the 2D RP–RP system is a highly versatile and powerful tool for many proteomics workflows.

Photo-crosslinking mass spectrometry enables the identification of protein-RNA interactions in living cells, pinpointing interaction interfaces at single-amino acid resolution. However, current isolation procedures for peptide-RNA crosslinks eliminate the RNA moiety, prohibiting sequencing of the RNA alongside the crosslinked peptide. Here, we introduce peptide-RNA crosslink isolation for sequencing by mass spectrometry or pepR-MS, a method that enriches peptide-RNA crosslinks with RNA chains of tunable length. Applied to breast cancer cells, pepR-MS identifies over 21,000 unique crosslinks at 4,757 crosslinking sites in 744 proteins. Employing different nucleases, we capture crosslinks with RNA moieties up to six nucleotides, revealing RNA crosslinking preferences at domain and subdomain resolution. Finally, we demonstrate mass spectrometry-based sequential sequencing of both peptide and RNA from the same crosslink, providing a starting point for the analysis of long-chain peptide-RNA crosslinks that map interaction interfaces across the proteome and transcriptome.

Three pioneering Oxford researchers are among the first recipients of the Royal Society Faraday Discovery Fellowships, prestigious long-term awards to support exceptional mid-career research leaders

Field Asymmetric Ion Mobility Spectrometry (FAIMS) enhances signal to noise ratio filtering ions based on their differential mobility, making it an indispensable tool for single-cell and low-input pro...

Encephalomyocarditis virus (EMCV) infects a range of species, causing economically important reproductive disorders in pigs and encephalitis and myocarditis in rodents. Due to its wide host range, it ...

Proteomic analyses offer insights into how KRAS inhibitors affect oncogenic KRAS signaling.