Immunoprecipitation on magnetic beads and liquid chromatography-tandem mass spectrometry for carbonic anhydrase II quantification in human serum.
In this study, a magnetic bead-based platform amenable to high-throughput protein carbonic anhydrase II (CA II) capture is presented. The key steps in this approach involved immunoaffinity purification of the target protein from serum followed by on-bead digestion with trypsin to release a surrogate peptide. This tryptic peptide was quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) operating in multiple reaction monitoring acquisition mode. Using a synthetic peptide standard and a structural analogue free-labeled internal standard, the resulting concentration was stoichiometrically converted to CA II serum concentration. The analytical steps, such as preparation of immunobeads, protein capture, proteolysis, and calibration, were optimized. The method was validated in terms of recovery (77%), reproducibility (relative standard deviation [RSD]<12%), and method detection limit (0.5 pmol ml(-1)). The developed method was applied to determining the CA II in eight healthy subjects, and the concentration measured was 27.3 pmol ml(-1) (RSD = 65%).
Callipo
Luciano
L
Department of Chemistry, Sapienza University of Rome, 00185 Roma, Italy.
Caruso
Giuseppe
G
Foglia
Patrizia
P
Gubbiotti
Riccardo
R
Samperi
Roberto
R
Laganà
Aldo
A
D000595:Amino Acid Sequence; D055910:Antibodies, Immobilized / Q000276:immunology / Q000378:metabolism; D024402:Carbonic Anhydrase II / Q000097:blood* / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D057166:High-Throughput Screening Assays; D006801:Humans; D047468:Immunoprecipitation; D008280:Magnetics; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D055910:Antibodies, Immobilized; D014357:Trypsin; D024402:Carbonic Anhydrase II