Multiplexed Proteomics of Brain Regions

Peptides were labeled with tandem-mass tag (TMT) isobaric labels (TMTpro 16plex™ Isobaric Label Reagent Set, Lot UL296296, Thermo Fisher Scientific). Ten micrograms of each sample were aliquoted into a final volume of 100 µL of 100 mM TEAB, and peptides were labeled according to the manufacturer’s protocol. Multiplexed samples were fractionated by high pH reversed-phase chromatography (1260 Infinity II, Agilent Technologies) as previously described [23 (link)]. Each multiplex contained a TMT channel of pooled samples specific for a given brain region, a channel of pooled samples representing all the brain regions and 14 channels corresponding to 14 individual patient samples. Twenty-four concatenated fractions were generated for global LC-MS/MS analysis.

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Teng P.N., Barakat W., Tran S.M., Tran Z.M., Bateman N.W., Conrads K.A., Wilson K.N., Oliver J., Gist G., Hood B.L., Zhou M., Maxwell G.L., Leggio L., Conrads T.P, & Lee M.R. (2023). Brain proteomic atlas of alcohol use disorder in adult males. Translational Psychiatry, 13, 318.

Publication 2023

TMTpro 16plex™ Isobaric Label Reagent Set, 1260 Infinity II

Brain Ms ms Patient Peptides Reversed phase chromatography Teab

Corresponding Organization : Veterans Health Administration

Other organizations : Henry M. Jackson Foundation, Inova Health System, National Institute on Alcohol Abuse and Alcoholism, National Institute on Drug Abuse

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Variable analysis

independent variables

Labeling with tandem-mass tag (TMT) isobaric labels (TMTpro 16plex™ Isobaric Label Reagent Set, Lot UL296296, Thermo Fisher Scientific)

dependent variables

Peptide abundance

control variables

Sample volume (10 micrograms of each sample aliquoted into a final volume of 100 µL of 100 mM TEAB)
Labeling protocol (peptides were labeled according to the manufacturer's protocol)
Fractionation method (high pH reversed-phase chromatography)
Mass spectrometry analysis (global LC-MS/MS analysis)

positive controls

A TMT channel of pooled samples specific for a given brain region

negative controls

A channel of pooled samples representing all the brain regions

Annotations

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