NIH_NitaLazar - Manes_BMDM_TLR-Chemotaxis_2021

Absolute Protein Quantitation of the Mouse Macrophage Toll-Like Receptor and Chemotaxis Pathways
Data License: CC BY 4.0 | ProteomeXchange: PXD031697 | doi: https://doi.org/10.6069/44s8-9f68
  • Organism: Mus musculus
  • Instrument: Q Exactive HF
  • SpikeIn: Yes
  • Keywords: macrophage, chemotaxis pathway, Toll-like receptor pathway, parallel reaction monitoring
  • Lab head: Aleksandra Nita-Lazar Submitter: Nathan Manes
Abstract
The Toll-like receptor and chemotaxis pathways are key components of the innate immune system. Computational modeling and simulation at the molecular interaction level can be used to study complex biological pathways, but protein concentration values must be input as model parameters. In this investigation, targeted mass spectrometry assays were developed and used to measure the absolute abundance (copies/cell) of proteins of the mouse macrophage Toll-like receptor 4 (TLR4) and chemotaxis pathways. The data produced by this investigation can be used for pathway modeling and simulation, as well as for other systems biology research.
Experiment Description
Scheduled parallel reaction monitoring (PRM) LC-MS assays were developed using an UltiMate 3000 nanoLC coupled to a Q Exactive HF (QEHF) mass spectrometer. Heavy isotope-labeled, purified, quantitated internal peptide standards (279 peptides total; 29 are phosphopeptides) were purchased for stable isotope dilution LC-QEHF-PRM assays to measure the absolute abundance of the target proteins. Similar work (LC-SRM) has been described previously <Manes et al 2015 Mol Cell Proteomics 14:2661-81><Manes et al 2015 J Vis Exp e52959>. Mouse bone marrow-derived macrophages (BMDMs) were prepared using a protocol very similar to two previously published protocols <Trouplin et al 2013 J Vis Exp e50966><Amend et al 2016 J Vis Exp e53936>. All procedures were approved by the NIAID Animal Care and Use Committee (NIH). The BMDM cells were prepared for LC-MS as described previously <Manes et al 2015 J Vis Exp e52959>. Each of the eleven preparations were enough for 50 LC-MS runs, and each preparation contained either 0 ug or 50 ug of digested BMDM protein, and each also contained 1 pmol of firefly luciferase. The eight preparations with 50 ug BMDMs were LC-PRM analyzed twice; the three samples with 0 ug BMDMs were LC-PRM analyzed once.
Sample Description
B0-1 = 50 fmol internal peptide stds. B0-2 = 500 fmol internal peptide stds. B0-3 = 5000 fmol internal peptide stds. B1-0 = 50 ug BMDM BioRep1. B1-1 = 50 ug BMDM BioRep1 + 50 fmol internal peptide stds. B1-2 = 50 ug BMDM BioRep1 + 500 fmol internal peptide stds. B1-3 = 50 ug BMDM BioRep1 + 5000 fmol internal peptide stds. B4-0 = 50 ug BMDM BioRep2. B4-1 = 50 ug BMDM BioRep2 + 50 fmol internal peptide stds. B4-2 = 50 ug BMDM BioRep2 + 500 fmol internal peptide stds. B4-3 = 50 ug BMDM BioRep2 + 5000 fmol internal peptide stds.
Created on 2/16/22, 12:11 AM
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Manes TLR Chemotaxis 2022.sky.zip2022-02-16 00:10:1372815957,45619