title: An integrated high-performance liquid chromatography-mass spectrometry system for the activity-dependent analysis of matrix metalloproteases.
abstract: Matrix metalloproteases (MMPs) comprise a family of enzymes that play important roles in mediating angiogenesis, the remodelling of tissues and in cancer metastasis. Consequently, they are attractive targets for therapeutic intervention in chronic inflammation, cancer and neurological disorders. In order to study MMPs in body fluids in an activity-dependent manner, we have developed an automated, integrated system comprising an immobilized inhibitor cartridge for activity-dependent enrichment, an immobilized trypsin reactor for rapid on-line proteolysis and a capillary or nanoLC-MS system for separation and identification of the obtained peptide fragments. This targeted proteomics system was optimized with respect to recovery and evaluated through the analysis of urine samples that were spiked with recombinant MMP-12. MMP-12 specific peptide fragments were easily detected in a nanoLC-MS analysis of 500 microL crude urine spiked at a level of 8 nM. These results show the feasibility of selective, activity-dependent enrichment of MMPs from a non-treated biofluid at low nM concentrations.
authors.affiliation: Analytical Biochemistry, Center for Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
authors.forename: Robert
authors.initials: R
authors.lastname: Freije
authors.forename: Theo
authors.initials: T
authors.lastname: Klein
authors.forename: Bert
authors.initials: B
authors.lastname: Ooms
authors.forename: Henk F
authors.initials: HF
authors.lastname: Kauffman
authors.forename: Rainer
authors.initials: R
authors.lastname: Bischoff
mesh_terms: D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D053508:Matrix Metalloproteinase 12 / Q000652:urine; D045726:Metalloproteases / Q000652:urine*; D040901:Proteomics / Q000379:methods
chemical_list: D045726:Metalloproteases; D053508:Matrix Metalloproteinase 12