High-throughput protein characterization using mass spectrometric immunoassay.
A high-throughput mass spectrometric immunoassay system for the analysis of proteins directly from plasma is reported. A 96-well format robotic workstation was used to prepare antibody-derivatized affinity pipette tips for subsequent use in the extraction of specific proteins from plasma and deposition onto 96-well format matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) targets. Samples from multiple individuals were screened with regard to the plasma protein transthyretin (TTR), followed by analysis of the same plasma samples for the transthyretin-associated transport protein, retinol-binding protein (RBP). Analyses were able to detect the presence of posttranslationally modified TTR and RBP, as well as a mutation present in the TTR of one individual. Subsequent analyses of wild-type and mutated TTR using enzymatically active MALDI-TOF MS targets were able to identify the site and nature of the point mutation. The approach represents a rapid (approximately 100 samples/2 h, reagent preparation-to-data) and accurate means of characterizing specific proteins present in large numbers of individuals for proteomic and clinical/diagnostic purposes.
Kiernan
Urban A
UA
Intrinsic Bioprobes, Inc., 625 S. Smith Road Suite, 22, Tempe, Arizona 85281, USA.
Tubbs
Kemmons A
KA
Gruber
Karl
K
Nedelkov
Dobrin
D
Niederkofler
Eric E
EE
Williams
Peter
P
Nelson
Randall W
RW
D000915:Antibody Affinity; D001798:Blood Proteins / Q000032:analysis; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008297:Male; D011228:Prealbumin / Q000032:analysis*; D012177:Retinol-Binding Proteins / Q000032:analysis*; D054839:Retinol-Binding Proteins, Plasma; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D001798:Blood Proteins; D011228:Prealbumin; D012177:Retinol-Binding Proteins; D054839:Retinol-Binding Proteins, Plasma