Digital Microfluidics and Magnetic Bead-Based Intact Proteoform Elution for Quantitative Top-down Nanoproteomics of Single C.†elegans Nematodes.

While most nanoproteomics approaches for the analysis of low-input samples are based on bottom-up proteomics workflows, top-down approaches enabling proteoform characterization are still underrepresented. Using mammalian cell proteomes, we established a facile one-pot sample preparation protocol based on protein aggregation on magnetic beads and intact proteoform elution using 40 % formic acid. Performed on a digital microfluidics device, the workflow enabled sensitive analyses of single Caenorhabditis elegans nematodes, thereby increasing the number of proteoform identifications compared to in-tube sample preparation by 46 %. Label-free quantification of single nematodes grown under different conditions allowed to identify changes in the abundance of proteoforms not distinguishable by bottom-up proteomics. The presented workflow will facilitate proteoform-directed analysis on samples of limited availability.

Digital Microfluidics Supported Microproteomics for Quantitative Proteome Analysis of Single Caenorhabditis elegans Nematodes.

Miniaturization of sample preparation, including omissible manual sample handling steps, is key for reproducible nanoproteomics, as material is often restricted to only hundreds of cells or single model organisms. Here, we demonstrate a highly sensitive digital microfluidics (DMF)-based sample preparation workflow making use of single-pot solid-phase enhanced sample preparation (SP3) in combination with high-field asymmetric-waveform ion mobility spectrometry (FAIMS), and fast and sensitive ion trap detection on an Orbitrap tribrid MS system. Compared to a manual in-tube SP3-supported sample preparation, the numbers of identified peptides and proteins were markedly increased, while lower standard deviations between replicates were observed. We repeatedly identified up to 5000 proteins from single nematodes. Moreover, label-free quantification of protein changes in single Caenorhabditis elegans treated with a heat stimulus yielded 45 differentially abundant proteins when compared to the untreated control, highlighting the potential of this technology for low-input proteomics studies. LC-MS data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD033143.

Cellular bicarbonate accumulation and vesicular proton transport promote calcification in the sea urchin larva.

The sea urchin embryo develops a calcitic endoskeleton through intracellular formation of amorphous calcium carbonate (ACC). Intracellular precipitation of ACC, requires [Formula: see text] concentrating as well as proton export mechanisms to promote calcification. These processes are of fundamental importance in biological mineralization, but remain largely unexplored. Here, we demonstrate that the calcifying primary mesenchyme cells (PMCs) use Na+/H+-exchange (NHE) mechanisms to control cellular pH homeostasis during maintenance of the skeleton. During skeleton re-calcification, pHi of PMCs is increased accompanied by substantial elevation in intracellular [Formula: see text] mediated by the [Formula: see text] cotransporter Sp_Slc4a10. However, PMCs lower their pHi regulatory capacities associated with a reduction in NHE activity. Live-cell imaging using green fluorescent protein reporter constructs in combination with intravesicular pH measurements demonstrated alkaline and acidic populations of vesicles in PMCs and extensive trafficking of large V-type H+-ATPase (VHA)-rich acidic vesicles in blastocoelar filopodial cells. Pharmacological and gene expression analyses underline a central role of the VHA isoforms Sp_ATP6V0a1, Sp_ATP6V01_1 and Sp_ATPa1-4 for the process of skeleton re-calcification. These results highlight novel pH regulatory strategies in calcifying cells of a marine species with important implications for our understanding of the mineralization process in times of rapid changes in oceanic pH.