timsTOF HT Improves Protein Identification and Quantitative Reproducibility for Deep Unbiased Plasma Protein Biomarker Discovery.

Mass spectrometry (MS) is a valuable tool for plasma proteome profiling and disease biomarker discovery. However, wide-ranging plasma protein concentrations, along with technical and biological variabilities, present significant challenges for deep and reproducible protein quantitation. Here, we evaluated the qualitative and quantitative performance of timsTOF HT and timsTOF Pro 2 mass spectrometers for analysis of neat plasma samples (unfractionated) and plasma samples processed using the Proteograph Product Suite (Proteograph) that enables robust deep proteomics sampling prior to mass spectrometry. Samples were evaluated across a wide range of peptide loading masses and liquid chromatography (LC) gradients. We observed up to a 76% increase in total plasma peptide precursors identified and a >2-fold boost in quantifiable plasma peptide precursors (CV < 20%) with timsTOF HT compared to Pro 2. Additionally, approximately 4.5 fold more plasma peptide precursors were detected by both timsTOF HT and timsTOF Pro 2 in the Proteograph analyzed plasma vs neat plasma. In an exploratory analysis of 20 late-stage lung cancer and 20 control plasma samples with the Proteograph, which were expected to exhibit distinct proteomes, an approximate 50% increase in total and statistically significant plasma peptide precursors (q < 0.05) was observed with timsTOF HT compared to Pro 2. Our data demonstrate the superior performance of timsTOF HT for identifying and quantifying differences between biologically diverse samples, allowing for improved disease biomarker discovery in large cohort studies. Moreover, researchers can leverage data sets from this study to optimize their liquid chromatography-mass spectrometry (LC-MS) workflows for plasma protein profiling and biomarker discovery. (ProteomeXchange identifier: PXD047854 and PXD047839).

Transcription-translation coupling: direct interactions of RNA polymerase with ribosomes and ribosomal subunits.

In prokaryotes, RNA polymerase and ribosomes can bind concurrently to the same RNA transcript, leading to the functional coupling of transcription and translation. The interactions between RNA polymerase and ribosomes are crucial for the coordination of transcription with translation. Here, we report that RNA polymerase directly binds ribosomes and isolated large and small ribosomal subunits. RNA polymerase and ribosomes form a one-to-one complex with a micromolar dissociation constant. The formation of the complex is modulated by the conformational and functional states of RNA polymerase and the ribosome. The binding interface on the large ribosomal subunit is buried by the small subunit during protein synthesis, whereas that on the small subunit remains solvent-accessible. The RNA polymerase binding site on the ribosome includes that of the isolated small ribosomal subunit. This direct interaction between RNA polymerase and ribosomes may contribute to the coupling of transcription to translation.

1H NMR Metabolic Profiling of Earthworm (Eisenia fetida) Coelomic Fluid, Coelomocytes, and Tissue: Identification of a New Metabolite-Malylglutamate.

Earthworm metabolism is recognized as a useful tool for monitoring environmental insults and measuring ecotoxicity, yet extensive earthworm metabolic profiling using 1H nuclear magnetic resonance (NMR) spectroscopy has been limited in scope. This study aims to expand the embedded metabolic material in earthworm coelomic fluid, coelomocytes, and tissue to aid systems toxicology research. Fifty-nine metabolites within Eisenia fetida were identified, with 47 detected in coelomic fluid, 41 in coelomocytes, and 54 in whole-worm samples and tissue extracts. The newly detected but known metabolites 2-aminobutyrate, nicotinurate, Nδ,Nδ,Nδ-trimethylornithine, and trigonelline are reported along with a novel compound, malylglutamate, elucidated using 2D NMR and high-resolution MS/MS. We postulate that malylglutamate acts as a glutamate/malate store, chelator, and anionic osmolyte and helps to provide electrolyte balance.

Cell and Protein Recognition at a Supported Bilayer Interface via In Situ Cavitand-Mediated Functional Polymer Growth.

Water-soluble deep cavitands embedded in a supported lipid bilayer are capable of anchoring ATRP initiator molecules for the in situ synthesis of primary amine-containing polymethacrylate patches at the water:membrane interface. These polymers can be derivatized in situ to incorporate fluorescent reporters, allow selective protein recognition, and can be applied to the immobilization of nonadherent cells at the bilayer interface.