TY - JOUR
T1 - 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
AU - Aivado, Manuel
AU - Spentzos, Dimitrios
AU - Alterovitz, Gil
AU - Otu, Hasan H.
AU - Grall, Franck
AU - Giagounidis, Aristoteles A.N.
AU - Wells, Meghan
AU - Cho, Je Yoel
AU - Germing, Ulrich
AU - Czibere, Akos
AU - Prall, Wolf C.
AU - Porter, Chris
AU - Ramoni, Marco F.
AU - Libermann, Towia A.
N1 - Funding Information:
We wish to thank Susan E. Hankinson from the Channing Laboratory, Harvard Medical School, and Brigham and Women’s Hospital, Boston, MA for her thoughtful comments and for providing blinded plasma samples. We are gratefully indebted to Bärbel Junge and Sabine Haase for their numerous serum sample preparations. This study was supported by National Institutes of Health Grants U24 DK 58739 and 1RO1 CA 85467 to TA L. MA was awarded a scholarship from the German Dr.-Mildred-Scheel Cancer Foundation, and GA’s work was supported by a fellowship from the Whitaker Foundation.
PY - 2005
Y1 - 2005
N2 - 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 26.7% to 32.6%, depending on the signal-to-noise ratio threshold used.
AB - 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 26.7% to 32.6%, depending on the signal-to-noise ratio threshold used.
KW - High-throughput
KW - Plasma protein profiling
KW - Proteomics
KW - Serum protein profiling
KW - Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI TOF-MS)
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U2 - 10.1515/CCLM.2005.022
DO - 10.1515/CCLM.2005.022
M3 - Article
C2 - 15843205
AN - SCOPUS:20144372083
SN - 1434-6621
VL - 43
SP - 133
EP - 140
JO - Clinical Chemistry and Laboratory Medicine
JF - Clinical Chemistry and Laboratory Medicine
IS - 2
ER -