title: Optimized conditions for a quantitative SELDI TOF MS protein assay.
abstract: The development of peptide/protein analyte assays for the purpose of diagnostic tests is driven by multiple factors, including sample availability, required throughput, and quantitative reproducibility. Laser Desorption/ionization mass spectrometry methods (LDI-MS) are particularly well suited for both peptide and protein characterization, and combining chromatographic surfaces directly onto the MS probe in the form of surface enhanced laser desorption/ionization (SELDI)-biochips has improved the reproducibility of analyte detection and provided effective relative quantitation. Here, we provide methods for developing reproducible SELDI-based assays by providing a complex artificial protein matrix background within the sample to be analyzed that allows for a common and reproducible ionization background as well as internal normalization standards. Using this approach, quantitative assays can be developed with CVs typically less than 10% across assays and days. Although the method has been extensively and successfully implemented in association with a protein matrix from E. coli, any other source for the complex protein matrix can be considered as long as it adheres to a set of conditions including the following: (1) the protein matrix must not provide interferences with the analyte to be detected, (2) the protein matrix must be sufficiently complex such that a majority of ion current generated from the desorption of the sample comes from the complex protein matrix, and (3) specific and well-resolved protein matrix peaks must be present within the mass range of the analyte of interest for appropriate normalization.
authors.affiliation: Laboratory Separations Division, Bio-Rad Laboratories, Hercules, CA, USA. lee_lomas@bio-rad.com
authors.forename: Lee
authors.initials: L
authors.lastname: Lomas
authors.forename: Charlotte H
authors.initials: CH
authors.lastname: Clarke
authors.forename: Vanitha
authors.initials: V
authors.lastname: Thulasiraman
authors.forename: Eric
authors.initials: E
authors.lastname: Fung
mesh_terms: D016477:Artifacts; D014408:Biomarkers, Tumor / Q000097:blood / Q000737:chemistry; D001774:Blood Chemical Analysis; D002138:Calibration; D003545:Cysteine; D005260:Female; D006801:Humans; D010051:Ovarian Neoplasms; D011228:Prealbumin / Q000032:analysis / Q000737:chemistry; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D020033:Protein Isoforms / Q000097:blood / Q000737:chemistry; D012015:Reference Standards; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods* / Q000592:standards
chemical_list: D014408:Biomarkers, Tumor; D011228:Prealbumin; D020033:Protein Isoforms; D003545:Cysteine