title: Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for monitoring the digestion of phosphatidylcholine by pancreatic phospholipase A(2).
abstract: Different methods were established for monitoring the phospholipase A(2)(PLA(2)) activity but all of them are rather cumbersome and time consuming. In this paper we have investigated the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the determination of the PLA(2) activity. Phosphatidylcholine (PC) was digested with pancreatic PLA(2) under different conditions, i.e., various Ca(2+), PC, and PLA(2) concentrations. The digestion products were analyzed by MALDI-TOF MS and the concentration of lysophosphatidylcholine (LPC)-generated upon PLA(2) digestion-was determined by the application of an internal standard (known concentration) and by a comparison of their signal-to-noise ratios. The results clearly demonstrate that the LPC concentration determined from the MALDI-TOF mass spectra correlates directly with the activity of the applied enzyme. Additionally, LPC concentration increased with an increase in Ca(2+), as well as in the PC concentration. A single MALDI-TOF mass spectrum provides immediate information on the digestion products as well as on the residual substrate without requirements for any previous derivatization. MALDI-TOF MS can be easily and simply applied for monitoring the PLA(2) activity and we assume that this method might also be useful for other types of phospholipases.
authors.affiliation: Institute of Medical Physics and Biophysics, Medical Faculty, University of Leipzig, Germany. petm@medizin.uni-leipzig.de
authors.forename: Marijana
authors.initials: M
authors.lastname: Petković
authors.forename: Julia
authors.initials: J
authors.lastname: Müller
authors.forename: Matthias
authors.initials: M
authors.lastname: Müller
authors.forename: Jürgen
authors.initials: J
authors.lastname: Schiller
authors.forename: Klaus
authors.initials: K
authors.lastname: Arnold
authors.forename: Jürgen
authors.initials: J
authors.lastname: Arnhold
mesh_terms: D000818:Animals; D002122:Calcium Chloride / Q000494:pharmacology; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000032:analysis; D010179:Pancreas / Q000201:enzymology*; D010713:Phosphatidylcholines / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013552:Swine
chemical_list: D008244:Lysophosphatidylcholines; D010713:Phosphatidylcholines; D010741:Phospholipases A; D002122:Calcium Chloride