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.
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)
UR - http://www.scopus.com/inward/record.url?scp=20144372083&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=20144372083&partnerID=8YFLogxK
U2 - 10.1515/CCLM.2005.022
DO - 10.1515/CCLM.2005.022
M3 - Article
C2 - 15843205
AN - SCOPUS:20144372083
VL - 43
SP - 133
EP - 140
JO - Clinical Chemistry and Laboratory Medicine
JF - Clinical Chemistry and Laboratory Medicine
SN - 1434-6621
IS - 2
ER -