Use of Edman degradation sequence analysis and matrix-assisted laser desorption/ionization mass spectrometry in designing substrates for matrix metalloproteinases

The matrix metalloproteinase (MMP) family has been implicated in the proces s of a variety of diseases such as arthritis, atherosclerosis, and tumor ce ll metastasis. We have been designing single-stranded peptides (SSPs) and t riple-helical peptides (THPs) as potential discriminatory MMP substrates. E dman degradation sequence and matrix-assisted laser desorption/ionization m ass spectrometric (MALDI-MS) analyses of proeolytic activity have been util ized to aid in further substrate design. THP models of the alpha 1(I)772-78 6 sequence from type I collagen were synthesized to examine the triple-heli cal substrate specificity of MMP family members. Sequence and MALDI-MS anal yses were used in conjunction with a fluorometric assay to determine the ex act point of cleavage by each MMP. MMP-1 (interstitial collagenase) cleaved the substrates at a single Gly-ne bond, analogous to the cleavage site in type I collagen. MMP-2 (M-r 72 000 type TV collagenase; gelatinase A) was f ound to cleave the substrates at two sites, a Gly-IIe bond and a Gly-GIn bo nd. MMP-3 (stromelysin 1) was found to cleave only one of the substrates;af ter reaction for 48 h. Ultimately, sequence and MALDI-MS analyses allowed u s to detect an additional cleavage site for MMP-2 in comparison to MMP-1, w hile MMP-3 was found to cleave a substrate after an extended time period. T he second cleavage site would cause the kinetic parameters for MMP-2 to be overestimated by the fluorometric assay. Further design variations for thes e substrates need to consider the presence of more stable triple-helical co nformation (to eliminate MMP-3 binding) and the removal of Gly-Gln bonds th at may be susceptible to MMP-2. (C) 2000 Elsevier Science B.V. All rights r eserved.