Electrospray ionization mass spectrometry: analysis of the Ca2+-binding properties of human recombinant alpha-parvalbumin and nine mutant proteins.
A set of 10 different recombinant human parvalbumins was used to establish a method for the investigation of the Ca2+-binding properties of proteins by electrospray ionization mass spectrometry (ESI-MS). Human PVWT was found to bind 2 mol Ca2+ ions/mol of protein, whereas its mutants (PVE101V, PVD90A, PVE62V, PVD51A, PVD90A,E101V, PVE62V,E101V, PVD51A,D90A, PVD51A,E62V, PVD51A,E62V, D90A,E101V) containing inactivating substitutions in the Ca2+-binding loops bind 0 or 1 Ca2+ 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 Ca2+-binding properties of rat and murine parvalbumins in muscle tissue extracts. ESI-MS revealed that (i) rat and murine alpha-parvalbumins each bind specifically two Ca2+ 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 Ca2+-binding proteins and other metalloproteins.
Troxler
H
H
Department of Pediatrics, Division of Clinical Chemistry and Biochemistry, University of Zurich, Steinwiesstrasse 75, Zurich, CH-8032, Switzerland.
Kuster
T
T
Rhyner
J A
JA
Gehrig
P
P
Heizmann
C W
CW
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D010320:Parvalbumins / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D010320:Parvalbumins; D011994:Recombinant Proteins; D002118:Calcium