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

Lauer-Fields, J; Nagase, H; Fields, G

Journal article

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

Abstract:: The matrix metalloproteinase (MMP) family has been implicated in the process of a variety of diseases such as arthritis, atherosclerosis, and tumor cell metastasis. We have been designing single-stranded peptides (SSPs) and triple-helical peptides (THPs) as potential discriminatory MMP substrates. Edman degradation sequence and matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analyses of proteolytic activity have been utilized to aid in further substrate design. THP models of the alpha1(I)772-786 sequence from type I collagen were synthesized to examine the triple-helical substrate specificity of MMP family members. Sequence and MALDI-MS analyses were used in conjunction with a fluorometric assay to determine the exact point of cleavage by each MMP. MMP-1 (interstitial collagenase) cleaved the substrates at a single Gly-Ile bond, analogous to the cleavage site in type I collagen. MMP-2 (Mr 72 000 type IV collagenase; gelatinase A) was found to cleave the substrates at two sites, a Gly-Ile bond and a Gly-Gln bond. MMP-3 (stromelysin 1) was found to cleave only one of the substrates after reaction for 48 h. Ultimately, sequence and MALDI-MS analyses allowed us to detect an additional cleavage site for MMP-2 in comparison to MMP-1, while MMP-3 was found to cleave a substrate after an extended time period. The second cleavage site would cause the kinetic parameters for MMP-2 to be overestimated by the fluorometric assay. Further design variations for these substrates need to consider the presence of more stable triple-helical conformation (to eliminate MMP-3 binding) and the removal of Gly-Gln bonds that may be susceptible to MMP-2.

Publication status:: Published

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APA Style

Lauer-Fields, J., Nagase, H., & Fields, G. (2000). Use of Edman degradation sequence analysis and matrix-assisted laser desorption/ionization mass spectrometry in designing substrates for matrix metalloproteinases. Journal of Chromatography. A, 890(1), 117–125.

MLA Style

Lauer-Fields, J, et al. “Use of Edman Degradation Sequence Analysis and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry in Designing Substrates for Matrix Metalloproteinases.” Journal of Chromatography. A, vol. 890, no. 1, 2000, pp. 117–25.

Chicago Style

Lauer-Fields, J, H Nagase, and G Fields. 2000. “Use of Edman Degradation Sequence Analysis and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry in Designing Substrates for Matrix Metalloproteinases.” Journal of Chromatography. A 890 (1): 117–25.
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