In situ AP/MALDI-MS characterization of anchored matrix metalloproteinases.
Several different procedures are available for the immobilization of proteins on solid supports, as many advantages derive from this approach, such as the possibility to develop new protein solid-state assays. Enzymes that are anchored on gold surfaces can interact with several different molecules in a tag-free environment, opening the way to surface plasmon resonance (SPR) investigations. Nevertheless, it is often important to know the identity of the affinity-retained analyte, and mass spectrometric analysis, via its unique molecular mass identification, represents a very valuable complementary method. There are many pieces of evidence to suggest that matrix metalloproteinases (MMPs) are involved in normal and pathological processes, including embryogenesis, wound healing, inflammation, arthritis and cancer, but presumably also exhibiting other functions. The search for new inhibitors of MMPs has prompted research towards the development of new solid-state assays for the rapid evaluation of MMP activity. We have already reported the possibility of measuring the activity of MMP-1 anchored on solid support by coupling SPR with ESI-MS analysis. In this work, we show the in situ atmospheric pressure (AP) MALDI-MS characterization of MMPs anchored on a gold chip with known surface coverage. The study extends the MS analysis to different proteins, and sequence coverage is reported for different digestion and MS procedures.
Grasso
Giuseppe
G
Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici, Via C. Ulpiani 27, Bari, Italy.
Fragai
Marco
M
Rizzarelli
Enrico
E
Spoto
Giuseppe
G
Yeo
Kwon Joo
KJ
D001274:Atmospheric Pressure; D006046:Gold; D006801:Humans; D020781:Matrix Metalloproteinase 1 / Q000032:analysis / Q000737:chemistry; D053508:Matrix Metalloproteinase 12 / Q000032:analysis / Q000737:chemistry; D020784:Matrix Metalloproteinase 8 / Q000032:analysis / Q000737:chemistry; D020782:Matrix Metalloproteinases / Q000032:analysis* / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D020349:Surface Plasmon Resonance; D014357:Trypsin
D006046:Gold; D014357:Trypsin; D020782:Matrix Metalloproteinases; D020784:Matrix Metalloproteinase 8; D053508:Matrix Metalloproteinase 12; D020781:Matrix Metalloproteinase 1