Cristea Lab - cGAS PTM

Identification of cGAS post-translational modifications

  • Organism: Homo sapiens
  • Instrument: Q Exactive HF
  • SpikeIn: No
  • Keywords: phosphorylation, acetylation, immune response, herpesvirus, IP-MS
  • Lab head: Ileana Cristea
DNA derived from the genetic material of pathogens or from cellular DNA damage provides a molecular pattern that can be sensed by pattern-recognition receptors of the mammalian innate immune system. In recent years, the cyclic GMP-AMP synthase (cGAS) protein has been characterized as a primary cytosolic DNA sensor during infection with bacteria, DNA viruses, or retroviruses. While the role of cGAS in downstream immune signaling through STING-TBK1-IRF3 proteins is well-defined, regulatory mechanisms of cGAS activity, such as through post-translational modifications (PTMs), are still an active area of research. Here, we report a comprehensive characterization of cGAS phosphorylations and acetylations in multiple cell types, HEK293T, primary human fibroblast, and THP-1 cells. A total of 11 PTMs (4 phosphorylations and 7 acetylations) were validated through a combination of data-dependent proteomic analysis and parallel reaction monitoring targeted MS on immunoaffinity purified cGAS. Of these, 3 phosphorylations and 5 acetylations have not been previously identified. The functions of these modifications were investigated by generating a series of mutants and measuring cGAS-dependent apoptotic and immune signaling activities.
Experiment Description
Two targeted mass spectrometry experiments were performed on immunoaffinity purified cGAS. First, 14 candidate cGAS PTM sites identified from discovery-based DDA experiments were targeted by parallel reaction monitoring for validation in three different cell types, HEK293T, primary human fibroblasts, and THP-1 cells, prepared from three independent biological replicates. Technical replicate injections were performed to cover all PTM sites. Second, the relative abundance of cGAS acetylation at Lys198 was quantified by PRM in uninfected and herpesvirus-infected human fibroblasts in three independent biological replicates.
Sample Description
Immunoaffinity purified cGAS was resolved by SDS-PAGE and processed by in-gel digestion. The region of the gel corresponding to either GFP-cGAS or untagged cGAS were excised and the in-gel digestion was performed using trypsin. Peptides were desalted using SDB-RPS stage tips, concentrated to near dryness by vacuum centrifugation, and resuspended in 1% formic acid (4 µl). PRM method development and analysis was performed using Skyline (ver. 19.1). Initial PRM methods were created with wide retention time scheduling windows (15 min) for 14 PTM peptides and two unmodified peptides in spectral libraries assembled from data-dependent search results. The initial PRM methods were used to analyze pooled peptides (2 µl) by nLC-PRM-MS on a Dionex Ultimate 3000 nanoRSLC HPLC connected online to a Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer equipped with an EASY-Spray ion source (Thermo Scientific). Peptides were separated by direct injection over an EASY-Spray PepMap C18 column (2 μm; 25 cm x 75 μm, Velos) for 60 min at 250 nl min-1 with a reverse phase linear ACN gradient from 3 to 30% solvent B (B: 97% ACN/0.1% formic acid/2.9% water, A: 0.1% formic/99.9% water). The mass spectrometer was configured for parallel reaction monitoring (PRM) acquisition. The detection of modified peptides was manually evaluated in Skyline. Refined scheduled PRM methods were generated with greater maximum injection time and narrower retention time windows, reducing the number of concurrent precursors to ≤ 3. Refined PRM methods were applied to biological replicates (N = 3) of uninfected HEK293T, HFF, and THP-1 cells. Validation of PTMs was performed by manual verification of peak picking. A candidate PTM was considered validated if it was detected ≥ 2 independent samples with ≥ 4 co-eluting fragment ions and received a dotp score > 0.70. For targeted quantification of acetyl-lysine 198 in uninfected and herpesvirus-infected fibroblast cells, the top three peak areas of the validated fragment ion chromatograms were summed, normalized by total cGAS levels using the unmodified peptide IQLEEYSNTR, and expressed relative to the average of the uninfected samples.
Created on 1/15/20, 10:44 PM
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20191205_cGAS PRM 06:25:531161720323
20191205_cGAS PRM 22:44:09133189