An integrated high-performance liquid chromatography–mass spectrometry system for the activity-dependent analysis of matrix metalloproteases

• Published in: Journal of Chromatography A Vol. 1189; no. 1; pp. 417 - 425
• Main Authors: Freije, Robert, Klein, Theo, Ooms, Bert, Kauffman, Henk F., Bischoff, Rainer
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Amsterdam Elsevier B.V 02.05.2008; Amsterdam; New York: Elsevier; Elsevier
• Subjects: Activity-based analysis; Affinity chromatography; Automated online analysis system; Biological and medical sciences; Chromatography, Affinity; Chromatography, High Pressure Liquid - methods; HPLC; Humans; Investigative techniques, diagnostic techniques (general aspects); Mass spectrometry; Mass Spectrometry - methods; Matrix Metalloproteinase 12 - urine; Medical sciences; Metalloprotease; Metalloproteases - urine; Miscellaneous. Technology; MMPs; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Proteomics; Proteomics - methods; Targeted proteomics; Targeted proteomics; Affinity chromatography; Proteomics; MMPs; Metalloprotease; Activity-based analysis; Mass spectrometry; Automated online analysis system; HPLC; Capillary column; Glomerulonephritis; Solid phase extraction; Biological fluid; Chemical analysis; On line; Peptides; HPLC chromatography; Macrophage elastase; Metalloendopeptidases; Electrospray; Chemical enrichment; Affinity adsorbent; Sample preparation; Proteolysis; Clinical biology; Kidney disease; Human; Urine; Automation; Urinary system disease; Serine endopeptidases; Enzyme; Coupled method; Enzyme inhibitor; Patient; Protein; Peptidases; Bioreactor; Trypsin; Analysis method; Peptide fragment; Hydrolases; Immobilized enzyme
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2007.10.059

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 μL 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.