October 28, 2014
Herein we demonstrate the efficacy of an unbiased proteomics screening approach for studying protein expression changes in the KC-Tie2 psoriasis mouse model, identifying multiple protein expression changes in the mouse and validating these changes in human psoriasis. KC-Tie2 mouse skin samples (n=3) were compared with littermate controls (n=3) using gel-based fractionation followed by label-free protein expression analysis. 5482 peptides mapping to 1281 proteins were identified and quantitated: 105 proteins exhibited fold-changes .2.0 including: stefin A1 (average fold change of 342.4; average P=0.0082; cystatin A, human orthologue); slc25a5 (average fold change of 46.2; average P=0.0318); serpinb3b (average fold change of 35.6 and an average P = 0.0345; serpinB1, human orthologue); and kallikrein related peptidase 6 (average fold change of 4.7; average P=0.2474; KLK6). We independently confirmed mouse gene expression-based increases of selected genes including serpinb3b (17.4-fold, P<0.0001), KLK6 (9.0-fold, P=0.002), stefin A1 (7.3-fold; P<0.001) and slc25A5 (1.5-fold; P=0.05) using qRT-PCR on a second cohort of animals (n=8). Parallel LC/MS/MS analyses on these same samples verified protein-level increases of 1.3-fold (slc25a5; P<0.05), 29,000-fold (stefinA1; P<0.01), 322-fold (KLK6; P<0.0001) between KC-Tie2 and control mice. To underscore the utility and translatability of our combined approach, we analyzed gene and protein expression levels in psoriasis patient skin and primary keratinocytes vs. healthy controls. Increases in gene expression for slc25a5 (1.8-fold), cystatin A (3.0-fold), KLK6 (5.8-fold) and serpinB1 (76-fold; all P < 0.05) were observed between healthy controls and involved lesional psoriasis skin and primary psoriasis keratinocytes. Moreover slc25a5, cystatin A, KLK6 and serpinB1 protein were all increased in lesional psoriasis skin compared to normal skin. These results highlight the usefulness of preclinical disease models using readily-available mouse skin and demonstrate the utility of proteomic approaches for identifying novel peptides/proteins that are differentially regulated in psoriasis that could serve as sources of auto-antigens or provide novel therapeutic targets for the development of new anti-psoriatic treatments.
Workflow summaries for the gel based label-free discovery and verification of
target proteins approaches.
Gel base label-free discovery workflow: Lysates from control and KC-Tie2 mice were separated by 1D SDS-PAGE. Eight gel regions per sample were excised resulting in 48 total regions. Each region was digested with Trypsin individually and analyzed for a total of 48 LC/MS/MS runs. Proteins and peptides were identified and quantified using Rosetta Elucidator software. This analysis yielded candidate proteins that were selected for verification (A). Verification of candidate proteins workflow: Tissue from 4 control and 4 KC-Tie2 mice was flash frozen in liquid nitrogen and then cryo-pulverized in preparation for verification of identified candidate proteins. The pulverized tissues were then subjected to FASP and analyzed individually by LC/MS/MS. Raw data files were searched using Mascot and target proteins were identified for verification. Peptides from elected candidate proteins were manually quantified and foldchanges were determined.
Results from: Lundberg KC, Fritz Y, Johnston A, Foster AM, Baliwag J, Gudjonsson JE, Schlatzer D, Gokulrangan G, McCormick TS, Chance MR, Ward NL. Mol Cell Proteomics. 2014 Oct 28. pii: mcp.M114.042242. [Epub ahead of print]