Russian Journal of Bioorganic Chemistry, Vol. 26, No. 9, 2000, р . 593

Quantitative Determination of Peptides and Proteins by MALDI MS

O. A. Mirgorodskaya*, ¹ Yu. P. Koz’min**, M. I. Titov**, N. V. Savel’eva*, R. Körner***, C. Sönksen***, A. I. Miroshnikov**, and P. Roepstorff***

*Institute of Cytology, Russian Academy of Sciences, Tikhoretskii pr. 4, St . Petersburg, 194064 Russia, **Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, GSP-7 Moscow , 117871 Russia , ***Department of Molecular Biology, University of Southern Denmark , Odense , DK-5230 Denmark

Abstract: A modified method of isotope dilution was applied to the quantitative determination of peptides and proteins by MALDI MS at subpicomolar level. The essence of the method consists in the quantitative analysis of the enzymic hydrolysis products rather than the starting compounds. This allows the measurements to be per-formed at a higher resolution and makes the method independent of the molecular mass of oligopeptides and proteins examined. Fragments obtained by hydrolysis of the same oligopeptide or protein in a known concentration by the same enzyme and labeled with the stable ¹⁸O isotope are used as internal standards. The label is introduced by carrying out the hydrolysis in H₂ ¹⁸O, and the oligopeptide concentration is calculated from the isotope distribution between the labeled and unlabeled hydrolysis products in the mass spectrum. This method was tested in the determination of concentrations of the angiotensinogen (1–14) fragment (oligopeptide), extracellular RNase from Bacillus amyloliquefaciens (protein) and its protein inhibitor, barstar M. Usefulness of this method in kinetic studies was also demonstrated.

Key words: peptides and proteins, quantitative determination; MALDI-MS; ¹⁸ O-labeled standards; enzymic hydrolysis; kinetic measurements