Dataset Information

FAIMS single cellUltrasensitive single-cell proteomics workflow identifies >1000 protein groups per mammalian cell

ABSTRACT: We report on the combination of nanodroplet sample preparation, ultra-low-flow nanoLC, high-field asymmetric ion mobility spectrometry (FAIMS), and the latest-generation Orbitrap Eclipse Tribrid mass spectrometer for greatly improved single-cell proteome coverage.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Brain, Hela Cell

SUBMITTER: Ryan Kelly

LAB HEAD: Ryan Kelly

PROVIDER: PXD019515 | Pride | 2020-11-24

REPOSITORIES: Pride

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			Action	DRS
	FAIMS_2CV_OTIT_HCD_300ITMS2_Blank_1.raw	Raw
	FAIMS_2CV_OTIT_HCD_300ITMS2_Blank_2.raw	Raw
	FAIMS_2CV_OTIT_HCD_300ITMS2_Blank_3.raw	Raw
	FAIMS_2CV_OTIT_HCD_300ITMS2_SingleInterNeuron_1.raw	Raw
	FAIMS_2CV_OTIT_HCD_300ITMS2_SingleInterNeuron_2.raw	Raw

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Project description:ALS is a uniformly fatal neurodegenerative disease in which motor neurons in the spinal cord and brain stem are selectively lost. Individual motor - groups of motor neurons innervating single muscles - show widely varying degrees of disease resistance: in the final stages of ALS, nearly all voluntary movement is lost but eye movement and eliminative and sexual functions remain relatively unimpaired. These functions are controlled by motor neurons of the oculomotor (III), trochlear (IV) and abducens (VI) nuclei in the midbrain and brainstem, and by Onuf's nucleus in the lumbosacral spinal cord, respectively. Correspondingly, in ALS autopsies the oculomotor and Onuf's nuclei are almost completely preserved. We used microarray profiling of isolated wildtype mouse motor neurons to identify genes whose expression was characteristic of both oculomotor and Onuf's nuclei but not of vulnerable lumbar spinal neurons, or vice versa. Three wild-type C57BL/6J P7 male animals were perfused with 30% sucrose, lumbosacral spinal cord and midbrain regions were rapidly recovered, embedded in OCT compound, and frozen in liquid nitrogen. 12 um-thick cryosections were mounted on RNAse-free, PEN-foil covered glass slides (Zeiss), fixed for 2 min in 100% EtOH, rinsed in 50% EtOH, stained with 1% cresyl violet for 2 min, rinsed with 50% EtOH, dehydrated in graded solutions of ethanol and air dried prior to LCM using PALM Microbeam system (Zeiss). From each animal, ~200 DL, L5, and oculomotor motor neurons were collected directly in lysis buffer. RNA was purified using Absolutely RNA, NanoPrep kit (Stratagene, La Jolla, CA). RNA integrity was assessed on the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). At least 1.5 ng of purified RNA was the starting material used in the WT-Ovation Pico RNA Amplification System (Nugen, San Carlos,CA) with the FL-Ovation cDNA Biotin Module V2 (Nugen) to generate labeled probe. 10 ug of biotinylated cRNA from three independent samples for each motor neuron group isolated by LCM was hybridized to on Affymetrix (Santa Clara, CA) Mouse Genome 430 2.0 Arrays. Gene ontology and pathway analysis was performed using DAVID Bioinformatics Resources 6.7 (National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD).

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FAIMS single cellUltrasensitive single-cell proteomics workflow identifies >1000 protein groups per mammalian cell

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