Electrospray Ionization Mass Spectrometry: Analysis of the Ca2+-Binding Properties of Human Recombinant α-Parvalbumin and Nine Mutant Proteins

✍ Scribed by H. Troxler; T. Kuster; J.A. Rhyner; P. Gehrig; C.W. Heizmann

• Publisher: Elsevier Science
• Year: 1999
• Tongue: English
• Weight: 90 KB
• Volume: 268
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1998.3015

✦ Synopsis

2؉ -binding loops bind 0 or 1 Ca 2؉ ion per protein molecule, depending on the degree of inactivation. These findings fully agree with previously reported results obtained by flow dialysis experiments. The RP-HPLC desalted metal-free proteins were analyzed in 10 mM ammonium acetate at pH 7.0. The experimental conditions were optimized with the recombinant parvalbumin test system before analyzing the Ca 2؉ -binding properties of rat and murine parvalbumins in muscle tissue extracts. ESI-MS revealed that (i) rat and murine ␣-parvalbumins each bind specifically two Ca 2؉ ions per protein molecule and (ii) both extracted parvalbumins were found to be posttranslationally modified; each protein is acetylated at the N-terminus. Finally, during our investigations of the murine parvalbumin a sequencing error was detected at the C-terminus where the amino acid at position 109 is Ser and not Thr as mentioned in the SwissProt data base (Accession No. P32848). This work demonstrates the great potential of the ESI-MS technique as a sensitive, specific, and rapid method for direct identification and determination of the stoichiometry of Ca 2؉ -binding proteins and other metalloproteins.