Altered signaling associated with chronic arsenic exposure in human skin keratinocytes
Modulation of signaling pathways upon chronic arsenic exposure remains poorly studied. Here we carried out SILAC-based quantitative phosphoproteomics analysis to dissect the signaling induced upon chronic arsenic exposure in human skin keratinocyte cell line, HaCaT. We identified 4,171 unique phosphosites derived from 2,000 proteins. We observed differential phosphorylation of 406 phosphosites (2-fold) corresponding to 305 proteins. Several pathways involved in cytoskeleton maintenance and organization were found to be significantly enriched (p<0.05). Our data revealed altered phosphorylation of proteins associated with adherens junction remodeling and actin polymerization. Kinases such as protein kinase C iota type (PRKCI), mitogen-activated protein kinase kinase kinase 1 (MAP3K1), tyrosine-protein kinase BAZ1B (BAZ1B) and STE20 like kinase (SLK) were found to be hyperphosphorylated. Our study provides novel insights into signaling perturbations associated with chronic arsenic exposure in human skin keratinocytes.
Sample Processing Protocol
HaCaT parental cells were adapted to DMEM supplemented with heavy stable isotopic forms of lysine and arginine (13C6 L-lysine and 13C6 L-arginine) for ten passages and the arsenic exposed cells were maintained in "light" media. Before harvesting, cells were washed with cold 1x PBS thrice and harvested in urea lysis buffer (20 mM HEPES (pH 8.0), 9 M urea, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate and 1 mM β-glycerophosphate), sonicated and centrifuged at 13,000 rpm at 15°C for 20 min. Equal amounts of protein from the two conditions were mixed and subjected to trypsin digestion. Briefly, the pooled lysates were reduced using DTT and alkylated with IAA followed by trypsin digestion. Trypsin was added in a ratio of 1:20 and incubated at 37°C for 16 hours. The reaction was stopped by acidifying the peptide solution with formic acid to a final concentration of 1%. The peptide sample was cleaned using Sep-Pak C18 cartridges (Waters, Milford, MA, USA) and lyophilized. The lyophilized peptide sample was subjected to basic pH reversed-phase liquid chromatography. A total of 90 fractions were collected, concatenated to 12 fractions and dried using speedvac for phosphopeptide enrichment. Peptide fractions were subjected to TiO2-based phosphopeptide enrichment. Briefly, TiO2 beads were incubated with DHB solution (80% ACN, 1% TFA, and 3% 2, 5-dihydroxybenzoic acid) for 1 h at room temperature. Each fraction was resuspended in 3% DHB solution and incubated with TiO2 beads at 1:1 ratio for 30 minutes at room temperature. Phosphopeptide-bound TiO2 beads were washed three times with DHB solution and twice with 40% ACN. Peptides were eluted three times with 40 μL of 2% ammonia solution into tubes containing 10 μL of 20% TFA on ice. The peptides were dried and resuspended in 30 µL of 0.1% TFA and desalted using C18 StageTips. The eluted peptides were vacuum dried and subjected to LC-MS/MS analysis. Twelve enriched phosphopeptide fractions from each of the two replicates were analyzed on LTQ Orbitrap Velos mass spectrometer interfaced with Easy-nLC II nanoflow liquid chromatography system as described earlier
Data Processing Protocol
Data were searched using Mascot (Version 2.2.0) and SEQUEST search algorithms against a RefSeq human protein database (Version 73) using Proteome Discoverer 2.0 software. The search parameters for both algorithms included: carbamidomethylation of cysteine as a fixed modification, oxidation of methionine, phosphorylation at serine, threonine and tyrosine (+79.966 Da) and SILAC labeling (13C6) at lysine and arginine as variable modifications (6.02013 Da). MS/MS spectra were searched with a precursor mass tolerance of 10 ppm and fragment mass tolerance of 0.05 Da. The data was searched against decoy database and the false discovery rate was set to 1% at the peptide level. The SILAC ratio for each phosphopeptide-spectrum match (phosphoPSM) was calculated by the quantitation node and the probability of phosphorylation for each site was calculated by the phosphoRS 3.1 node in the Proteome Discoverer. Phosphopeptides with > 75% localization probability at particular residue were selected for further analysis.
Mir SA, Renuse S, Sathe G, Khan AA, Patil AH, Nanjappa V, Bhat FA, Prasad TSK, Giri AK, Chatterjee A, Gowda H. Altered signaling associated with chronic arsenic exposure in human skin keratinocytes. Proteomics Clin Appl. 2017 Jul 21 PubMed: 28731282