Aebersold - GRB2 interactome by AP-SWATH

Robust temporal profiling of GRB2 protein complexes in primary T cells using SWATH mass spectrometry

  • Organism: Mouse
  • Instrument: 5600 Triple-TOF
  • SpikeIn: No
Spatiotemporal organization of protein interactions in cell signaling is a fundamental process that drives cellular functions. In this study, we present a method that combines mouse genetics and affinity purification (AP)-SWATH mass spectrometry — an advanced mass spectrometry workflow — to quantify high-confidence time-resolved GRB2 protein interactions in primary mouse T cells. Our approach provides a proof of concept that rapid, precise and reproducible quantitative measurements of protein interaction dynamics can be achieved in primary cells isolated from mammalian tissues. This method could be further applied to expedite the robust profiling of dynamic signaling complexes for key hub proteins in a range of cell and tissue types in vivo to resolve the tissue specific context of cell signaling events.
Experiment Description
In this study, we assessed whether affinity purification (AP)-SWATH — an advanced mass spectrometry-based workflow — could enable rapid, reliable and precise quantitative analysis of protein interaction dynamics from primary cells. Specifically, we generated a line of gene-targeted mice expressing a One-STrEP-tag (OST) at the carboxyl-terminus of endogenous GRB2 proteins. We isolated CD4+ T cells from GRB2-OST mice, lysed the cells before or at various times after activation with anti-CD3 plus anti-CD4 and then isolated protein complexes containing GRB2-OST through the use of Strep-Tactin-Sepharose beads (n=4). Samples were acquired in a SWATH-compatible 5600 Triple-TOF mass spectrometer. AP-SWATH data were analyzed using OpenSWATH and Skyline.
Sample Description
Short-term expanded CD4+ T cells (100 x 106) from GRB2OST and wild-type mice were left unstimulated or stimulated at 37°C with antibodies. In the last instance, CD4+ T cells were incubated with anti-CD3 (0.2 μg per 106 cells; 145-2C11; Exbio) and anti-CD4 (0.2 μg per 106 cells; GK1.5; Exbio) on ice, followed by one round of washing at 4°C. Cells were then incubated at 37°C for 5 min and subsequently left unstimulated or stimulated at 37°C with a purified Rabbit anti-Rat (0.4 μg per 106 cells; Jackson Immunoresearch) for 0.5, 2, 5 or 10 minutes at 37°C. Stimulation was stopped by the addition of a twice concentrated lysis buffer (100 mM Tris, pH 7.5, 270 mM NaCl, 1 mM EDTA, 20% glycerol, 0.2% n-dodecyl-β-maltoside) supplemented with protease and phosphatase inhibitors. After 10 min of incubation on ice, cell lysates were centrifuged at 14,000 rpm for 5 min at 4°C. Equal amount of post-nuclear lysates were incubated with prewashed Strep-Tactin Sepharose beads (IBA GmbH) for 1.5 h at 4°C on a rotary wheel. Beads were then washed 5 times with 1 ml of lysis buffer in the absence of detergent and of protease and phosphatase inhibitors. Proteins were eluted from the Strep-Tactin Sepharose beads with 2.5 mM D-biotin. For removal of D-biotin, samples were precipitated by addition of trichloroacetic acid (100%) to 25% (v/v) and incubation on ice for 1 h. Protein were pelleted by centrifugation at 13,000 rpm for 15 min at 4 ºC. Protein pellets were then washed 3 times with 200 µL ice-cold acetone with 5-min interspersed centrifugation. Washed protein pellets were dried by vacuum centrifugation at 45 ºC for 5 min and then resuspended in 25 µL 6 M urea, 50 mM NH4HCO3. Samples were diluted to 0.5 M urea with 50 mM NH4HCO3 before cysteines reduction (5 mM TCEP, 30 min at 37 ºC) and alkylation (10 mM iodoacetamide, 30 min at 37 ºC in the dark). Protein was digested overnight at 37 ºC by addition of 1 µg trypsin (2.5 uL Promega, sequence-grade, V5113). Trifluoroacetic acid (50%) was added to 1 % (v/v) to stop the reaction, and peptides were purified using C18 microspin columns (3 – 30 ug, Nest Group) and resuspended in 15 µL Buffer A (acetonitrile 2%, formic acid 0.1 %) containing iRT peptides for retention-time alignment (Biognosys). 4 µL of resuspended peptides was injected serially in SWATH and shotgun acquisition modes.
Created on 6/8/16, 6:35 AM
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