Abstract
Purpose: :
Characterization of MT1-MMP enzyme regulated protein expression in cornea fibroblast cells
Methods: :
Cornea fibroblast cells with partial deletion of the enzymatic domain of MT1-MMP or total MT1-MMP knock-out were generated and cultured. Nuclear proteins were isolated and compared using SDS-PAGE. Patterns of nuclear protein expressions were developed by coomassie blue staining. Three different protein bands separated by SDS-PAGE were prepared for mass spectrometry by reduction with DTT, alkylation with chloroacetamide followed by overnight incubation with trypsin (Promega). Peptides were extracted using ammonium bicarbonate followed by 50% acetonitrile in ammonium bicarbonate. They were concentrated by speedvac and separated by nano LC/MS/MS using a chip based HPLC system (Agilent Chip Cube) adapted to run on the Thermo LTQ-FT Ultra. Tandem mass spectra were extracted by Readw.exe (Institute for Systems Biology) version 4.3.1. Charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; Mascot version 2.2). Scaffold (version Scaffold 3.0.9, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications.
Results: :
Eighteen proteins were identified at ~170 kDa from total knock-out cells, 37 proteins from WT, and 29 proteins from enzymatic domain deleted cells. Identified proteins were categorized as function related cellular process, immune system, adhesion, or regulation of biological process. Proteins were also characterized by transcription regulator, enzyme, structure, and catalytic activity. Myosin-9, Filamin-A, DNA topoisomerase 2 alpha and beta, and DNA (cytosine-5)-methyltransferase 1 proteins were identified on MT1-MMP knock-out cells. Also, collagen Type I and III as a substrate of MT1-MMP proteins were identified on MT1-MMP knock-out cells. Ras GTPase-activating protein-binding protein 1 was identified from WT cells.
Conclusions: :
MT1-MMP regulation of extracellular matrix remodeling may be mediated, in part, by regulating different protein expression, including collagen Type I, cytoskeletal proteins and Ras GTPase-activating protein-binding protein 1.
Keywords: cornea: basic science • cornea: stroma and keratocytes • proteomics