Our general quantitative proteomics approach has been previously described (Phillips et al., 2015 (link); Titz et al., 2014 (link)) and the reader is referred to our main study publications for a high-level summary of the proteomics results (Phillips et al., 2015 (link) and Phillips et al., 2015b ). For convenience, we have summarized the experimental details in the Supplementary Methods . Briefly, samples were homogenized in tissue lysis buffer, proteins were precipitated, and 50 µg protein from each sample was used for further analysis. In the C57BL/6 study, samples were processed using iTRAQ 8-plex labeling (AB Sciex, Framingham, Massachusetts). For the Apoe−/− study, samples were processed using TMT 6-plex labeling (Thermo Scientific, Waltham, Massachusetts). Each procedure was performed according to the manufacturer’s instructions. Samples were analyzed by liquid chromatography coupled with tandem mass spectrometry using an Easy-nanoLC 1000 instrument connected online to a Q-Exactive MS mass-analyzer (Thermo Scientific). Data were normalized, quantified, and used to identify differentially abundant proteins in the R environment. The Benjamini–Hochberg FDR multiple test correction was employed and proteins with an adjusted P-value <.05 were considered to be differentially expressed.
Tmt 6 plex labeling
Manufactured by Thermo Fisher Scientific
TMT 6-plex labeling is a tool used for quantitative proteomics analysis. It enables simultaneous identification and relative quantification of proteins across multiple samples. The core function of this product is to facilitate multiplexed peptide and protein analysis through isobaric labeling.
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3 protocols using tmt 6 plex labeling
1
Quantitative Proteomics Workflow Summary
2
Quantitative Proteomics Workflow using TMT
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Dried peptides were dissolved in 80 L of 100 mM TEAB and quantified via BCA assay. Samples (10 μg) were supplemented with 150 fmol of internal standard (yeast ADH1, Waters) and subjected to labeling with TMT 6plex labeling (Thermo Fisher Scientific) following the manufacturer’s protocol. TMT-labeled samples were pooled, dried by lyophilization, and redissolved in 200 mM NH4HCO2, pH 10. Samples were fractionated under high pH reversed phase conditions on three layers of SDB-XC extraction disks (3 M, 1.07 mm × 0.50 mm i.d.) in 200 μL pipette tips into 60 μL volumes of 5,10, 15, 20, 22.5, 27.5, and 80% CH3CN in 200 mM NH4HCOO pH 10. The first two fractions were combined. The samples were dried and redissolved in 30 μL of 0.1% HCO2H in water and stored at − 80 °C until LC–MS analysis.
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Proteomic Analysis of Plasma Samples
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Plasma samples (6 time points/animal) were first processed in BSL-3 or BSL-4 containment by adding 25 µL SDS-PAGE solubilizing/reducing buffer to 75 µL sample and heating to 95 °C for 10 min. Samples were then removed from containment and stored at − 80 °C until processed by the iFASP method [28 (link)]. Briefly, 5 µL of each inactivated plasma sample was added to 200 µL 8 M Urea/100 mM Tris–HCL pH 8.5 (Solution UT8) and filtered through a Microcon-30 kDa Centrifugal Filter Unit with an Ultracel-30 membrane (Millipore, MRCF0R030) at 14,000 × G for 15 min. Following several washing steps with 100 mM Tris pH 8.0, proteins were alkylated with 55 mM Iodoacetamide and digested with 4 µg Trypsin/Lys-C (Promega, V5071) overnight at 37 °C. TMT 6-Plex labeling (Thermo Fisher, 90061) was performed directly on the FASP filters per the manufacturer’s instructions. All 6 single labelled samples were then combined at an equal volume, purified by C18 spin column, dried to completion by speed-vac and stored at − 20 °C until analyzed by LC MS/MS.
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