Valenta, A. | bioRxiv Science

bioRxiv2023

A high-sensitivity low-nanoflow LC-MS configuration for high-throughput sample-limited proteomics

This study demonstrates how optimized liquid chromatography-mass spectrometry (LC-MS) conditions combined with a 50 {micro}m internal diameter (I.D.) column operated at 100 nL/min enables high-sensitivity and high-throughput sample-limited proteomics analysis, including single-cell proteomics (SCP). Through systematic parameter evaluation, an optimized method was developed, capable of identifying [~]3,000 protein groups from 250 pg of HeLa protein digest using a 10-min gradient in the direct injection workflow using data-independent acquisition (DIA) from a library-free search method. Further improvements reduced the cycle time from 20 to 14.4 min by employing a trap-and-elute workflow, permitting 100 runs/day with 70% MS utilization. A proof of principle study indicated that ca. 1,700 protein groups were identified from single-cell samples without a library for label-free quantification (LFQ). In conclusion, we demonstrate a high-sensitivity LC-MS/MS configuration that serves the needs for limited sample analysis, permitting 100 runs/day throughout.

biochemistry↗

bioRxiv2020

Synaptotagmin-7 is a calcium sensor for facilitation at the splanchnic-chromaffin cell synapse

Disturbances that threaten homeostasis elicit activation of the sympathetic nervous system (SNS) and the adrenal medulla. The effectors discharge as a unit to drive global and immediate changes in whole-body physiology. Descending sympathetic information is conveyed to the adrenal medulla via preganglionic splanchnic fibers. These fibers pass into the gland and synapse onto chromaffin cells, which synthesize, store, and secrete catecholamines and vasoactive peptides. While the importance of the sympatho-adrenal branch of the autonomic nervous system has been appreciated for many decades, the mechanisms underlying transmission between presynaptic splanchnic neurons and postsynaptic chromaffin cells have remained obscure. In contrast to chromaffin cells, which have enjoyed sustained attention as a model system for exocytosis, even the Ca2+ sensors that are expressed within splanchnic terminals have not yet been identified. This study shows that a ubiquitous Ca2+-binding protein, synaptotagmin-7 (Syt7), is expressed within the fibers that innervate the adrenal medulla, and that its absence can alter synaptic transmission in the preganglionic terminals of chromaffin cells. The prevailing impact in presynapses that lack Syt7 is a decrease in synaptic strength and neuronal short-term plasticity. Evoked excitatory postsynaptic currents (EPSCs) in Syt7 KO preganglionic terminals are smaller in amplitude than in wild-type synapses stimulated in an identical manner. Splanchnic inputs also display robust short-term presynaptic facilitation, which is compromised in the absence of Syt7. These data reveal, for the first time, a role for any synaptotagmin at the splanchnic-chromaffin cell synapse. They also suggest that Syt7 has actions at synaptic terminals that are conserved across central and peripheral branches of the nervous system.

neuroscience↗