Carr - SigPath_pSTY_TSQQuantiva

Highly multiplexed quantitative phosphosite assay for biology and preclinical studies
Data License: CC BY 4.0 | ProteomeXchange: PXD026939 | doi: https://doi.org/10.6069/hjyt-z904
  • Organism: Homo sapiens
  • Instrument: TSQ Quantiva
  • SpikeIn: Yes
  • Keywords: Proteomics, post-translational modifications, targeted mass spectrometry, multiple reaction monitoring, selected reaction monitoring, breast cancer, medulloblastoma, CPTAC
  • Lab head: Carr Submitter: Hasmik Keshishian
Abstract
Reliable methods to quantify dynamic signaling changes across diverse pathways are needed to better understand the effects of disease and drug treatment in cells and tissues but are presently lacking. Here we present SigPath, a targeted mass spectrometry (MS) assay that measures 284 phosphosites in 200 phosphoproteins of biological interest. SigPath probes a broad swath of signaling biology with high throughput and quantitative precision. We applied the assay to investigate changes in phospho-signaling in drug-treated cancer cell lines, breast cancer preclinical models and human medulloblastoma tumors. In addition to validating previous findings, SigPath detected and quantified a large number of differentially regulated phosphosites newly associated with disease models and human tumors at baseline or with drug perturbation. Our results highlight the potential of SigPath to monitor phosphoproteomic signaling events and to nominate mechanistic hypotheses regarding oncogenesis, response and resistance to therapy.
Experiment Description
Multiple reaction monitoring (MRM) assay configuration using heavy labeled synthetic peptides was done on TSQ Quantiva triple quadrupole mass spectrometer (Thermo Fisher) coupled with Easy-nLC 1200 ultra-high pressure liquid chromatography (UPLC) system (Thermo Fisher) in several batches of 50 to 100 peptides each. Skyline Targeted Mass Spec Environment was used throughout assay configuration and all data analysis. First, spectral libraries for the peptides were generated on a Q Exactive mass spectrometer. Spectral libraries were uploaded to Skyline and 5-10 most intense fragment ions (transitions) for each peptide were selected for MRM assay configuration. Transitions containing phosphosite or helping with the assignment of the phosphosite were included in the transition list for assay configuration. Next, collision energies (CE) were optimized for all the transitions and peptides by liquid chromatography – multiple reaction monitoring mass spectrometry (LC-MRM/MS) on TSQ Quantiva using Skyline’s CE optimization module. For every transition starting with the instrument specific calculated CE tested 10 additional CEs (5 below and 5 above the calculated CE) in increments of 2. The list of transitions with varying CE values was exported from Skyline and used for building the MRM method in Xcalibur software. Equimolar mixture of peptides at 50fm/l was analyzed by LC-MRM/MS on Quantiva using this method. Resulting data was analyzed on Skyline which then selected the CE that resulted in the highest peak area for each transition. In the final step of CE optimization MRM data was acquired with optimized CE values for every transition. Using this dataset in Skyline, manually selected the best 3 to 6 transitions for every peptide giving highest priority to fragment ions of y-series with mass to charge (m/z) above the precursor, and ions containing phosphosite or helping with the site localization. For the peptides where the options were more limited also included ions of y-series with m/z below the precursor and b-series. While we configured the assay on TSQ Quantiva MS, other triple quadrupole instruments can be used for this assay with further optimization of MS specific parameters for each instrument (Abbatiello et al, 2015; Kuhn et al, 2012). After the CE optimization compiled 2 LC- MRM/MS methods; one for peptides enriched by IMAC strategy (231 IMAC and 50 IMACpY mixtures) and the second for peptides enriched by pY antibody strategy (71 pY and 50 IMACpY mixtures). Liquid chromatography was performed on 75m ID picofrit columns packed in-house to a length of 28-30cm with Reprosil C18-AQ 1.9m beads (Dr Maisch GmbH) with solvent A of 0.1% formic acid (FA) / 3% acetonitrile (ACN) and solvent B of 0.1% FA / 90% ACN at 200nL/min flow rate. Below are the details of the IMAC and pY LC-MRM/MS methods: IMAC LC-MRM/MS method: method duration – 160min, gradient – 2 to 6% solvent B in 1min, 6 to 30% B in 124min, 30 to 60% B in 9min, 60 to 90% B in 1min, followed by a hold at 90% B for 5min, and subsequent hold at 50% B for 19min. MS parameters include 3sec cycle time, Q1 and Q3 resolution of 0.4 and 0.7, respectively, retention time (RT) scheduling window of 10min. pY LC-MRM/MS method: method duration – 120min, gradient – 2 to 6% solvent B in 1min, 6 to 30% B in 84min, 30 to 60% B in 9min, 60 to 90% B in 1min, followed by a hold at 90% B for 5min, and subsequent hold at 50% B for 19min. MS parameters include 1.5 sec cycle time, Q1 and Q3 resolution of 0.4 and 0.7, respectively, RT scheduling window of 10min
Sample Description
Titration curve experiment: Five cell lines (OVCAR, Meljuso, H3122, PC9, A375) Assay evaluation: Ten cell lines (PC9, H3122, TMD8, Mino, PC3, OVCAR4, WM266.4, Mel-juso, A375, and RT112) Cell line perturbagen experiments H3122 and Ls513 cells treated with either DMSO or drug for 6 hours and 24hours Breast cancer xenograft (PDX) experiment: tissue from six models selected from Washington University human to mouse (WHIM) PDX collection (Mundt et al., 2018) (4, 30, 21, 6, 2, and 12). Medulloblastoma experiment: 39 tissue samples from medulloblastoma patients belonging to all 4 groups (Sonic Hedgehog (SHH), group 3 (GR3), group 4 (GR5) and WNT)
Created on 9/21/21, 8:44 PM