[Robotics and standardized sample drying time increase reproducibility]
>>Clin Chem Lab Med. 2005 Apr;43(2):133-140.
Optimization and evaluation of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) with reversed-phase protein arrays for protein profiling.
Aivado M, Spentzos D, Alterovitz G, Otu HH, Grall F, Giagounidis AA, Wells M, Cho JY, Germing U, Czibere A, Prall WC, Porter C, Ramoni MF, Libermann TA.
BIDMC Genomics Center and Bioinformatics Core, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA, Dana-Farber/Harvard Cancer Center Cancer Proteomics Core, Harvard Medical School, Boston, MA, USA and Department of Hematology, Oncology and Clinical Immunology, University of Dusseldorf, Dusseldorf, Germany.
Surface-enhanced laser desorption/ionization (SELDI) time-of-flight mass spectrometry with protein arrays has facilitated the discovery of disease-specific protein profiles in serum. Such results raise hopes that protein profiles may become a powerful diagnostic tool. To this end, reliable and reproducible protein profiles need to be generated from many samples, accurate mass peak heights are necessary, and the experimental variation of the profiles must be known. We adapted the entire processing of protein arrays to a robotics system, thus improving the intra-assay coefficients of variation (CVs) from 45.1% to 27.8% (p<0.001). In addition, we assessed up to 16 technical replicates, and demonstrated that analysis of 2-4 replicates significantly increases the reliability of the protein profiles. A recent report on limited long-term reproducibility seemed to concord with our initial inter-assay CVs, which varied widely and reached up to 56.7%. However, we discovered that the inter-assay CV is strongly dependent on the drying time before application of the matrix molecule. Therefore, we devised a standardized drying process and demonstrated that our optimized SELDI procedure generates reliable and long-term reproducible protein profiles with CVs ranging from 25.7% to 32.6%, depending on the signal-to-noise ratio threshold used.<<
Cheers, Tuck |
