Use of LC-MS/MS to Identify Matrilysin/Matrix Metalloproteinase-7(MMP7) Cleavage Products of Perlecan (PLN)/Heparan Sulfate Proteoglycan (HSPG2)

• Published in: Journal of biomolecular techniques 2013-05, Vol.24 (Suppl), p.S60-S60
• Main Authors: Grindel, B.J., Chung, L.W., Farach-Carson, M.C., Pennington, Christopher
• Format: Article
• Language: English
• Subjects: Poster Session Abstracts
• ISSN: 1524-0215; 1943-4731

Introduction: PLN/HSPG2, a 450 KDa heparan sulfate (HS) proteoglycan, is expressed in the basement membrane (BM) underlying epithelial and endothelial cells. Break down of PLN and other BM proteins is thought to play a critical role in prostate cancer (PCa) metastasis. During PCa metastasis, a variety of proteolytic enzymes are expressed. However, proteases associated with PCa, that can cleave PLN, have yet to be identified. An interesting extracellular protease upregulated in PCa invasion, whose ability to cleave PLN has not been investigated, is matrilysin (MMP-7). As part of a broader investigation LC-MS/MS analysis was used to identify cleavage products from MMP-7 PLN domain (DM)-IV3 digests. Experimental Methods: DM-IV of PLN was recreated recombinantly and purified as three separate pieces (IV1, IV2, IV3). DM-IV3, a 75 KDa PLN fragment, was incubated with MMP-7 and then subjected to LC-MS/MS analysis on a Thermo-Fisher LTQ-Orbitrap MS. The MS was operated in the ESI(+) mode using data dependent MS/MS scanning. Results: A total of 39 MMP-7 PLN DM-IV3 peptide fragments were identified by LC-MS/MS. They ranged in mass from 722 to 2475 Da with parent mass errors ranging from 0.7 to 3.9 ppm. Peptides were first identified by performing a “no-enzyme” MASCOT search on a small PLN database containing DM-IV3. MASCOT de novo sequencing and sequencing via mMass was also employed to confirm the identity of PLN peptide fragments. Seven larger peptides ranging in mass from 3827 Da to 10855 Da were also observed. LC-MS data indicates MMP-7 has a strong preference for cleaving aliphatic amino acids (L, I, V, A) at the P1? site. PLN DM-IV3 sequence coverage was 49% for the 39 identified peptides. Conclusion: LC-MS/MS data indicates that PLN DM-IV3 is cleaved by MMP-7 with fragments ranging in mass from 722 to 10855 Da being produced.