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TMT10 controlled mixtures - SILAC HeLa UPS1, acquired using SPS-MS3

ABSTRACT: The controlled mixtures were prepared to evaluate the ability of our proposed approach when dealing with the complicated design, e.g. multiple TMT mixtures with technical replicates. 500, 333, 250, and 62.5 fmol UPS1 peptides were spiked-into 50 g SILAC HeLa peptides in duplicate. It produced a dilution series corresponding to 1, 0.667, 0.5, and 0.125 of the highest UPS1 peptide amount (500 fmol). In addition, a reference sample was generated by pooling all four diluted UPS1 peptide samples (286.5 fmol) and combined with 50 g of SILAC HeLa in duplicate. These 10 replicates were labeled with TMT10-plex reagents and mixed together to pass LC-MS/MS analysis. The procedure was repeated to generate a total of five such controlled mixtures. To assess technical variability, three technical replicates were prepared for each mixture. Totally there are 15 MS runs from 5 TMT mixtures. LC-MS/MS was performed using an EASY-nLC 1200 ultrahigh pressure liquid chromatography (UHPLC) connected to an Orbitrap Fusion Lumos Tribrid and equipped with an EASY-spray source (Thermo Fisher Scientific, San Jose, CA). The data was acquired using an MS2/MS3 (also called multinotch MS3 or Synchronous Precursor Selection, SPS).

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Olga Vitek Tom Dunkley

PROVIDER: MSV000084264 | MassIVE | Fri Aug 30 15:46:00 BST 2019

SECONDARY ACCESSION(S): PXD015258

REPOSITORIES: MassIVE

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TMT10 controlled mixtures - SILAC HeLa UPS1, acquired using MS2-only strategy

Project description:The controlled mixtures were prepared to evaluate the ability of our proposed approach when dealing with the complicated design, e.g. multiple TMT mixtures with technical replicates. 500, 333, 250, and 62.5 fmol UPS1 peptides were spiked-into 50 g SILAC HeLa peptides in duplicate. It produced a dilution series corresponding to 1, 0.667, 0.5, and 0.125 of the highest UPS1 peptide amount (500 fmol). In addition, a reference sample was generated by pooling all four diluted UPS1 peptide samples (286.5 fmol) and combined with 50 g of SILAC HeLa in duplicate. These 10 replicates were labeled with TMT10-plex reagents and mixed together to pass LC-MS/MS analysis. The procedure was repeated to generate a total of five such controlled mixtures. To assess technical variability, three technical replicates were prepared for each mixture. Totally there are 15 MS runs from 5 TMT mixtures. LC-MS/MS was performed using an EASY-nLC 1200 ultrahigh pressure liquid chromatography (UHPLC) connected to an Orbitrap Fusion Lumos Tribrid and equipped with an EASY-spray source (Thermo Fisher Scientific, San Jose, CA). The same samples for MSV000084264 (or PXD0015258) are used. The data were acquired using MS2-only strategies.

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