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.
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.
Aivado
Manuel
M
BIDMC Genomics Center and Bioinformatics Core, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA.
Spentzos
Dimitrios
D
Alterovitz
Gil
G
Otu
Hasan H
HH
Grall
Franck
F
Giagounidis
Aristoteles A N
AA
Wells
Meghan
M
Cho
Je-Yoel
JY
Germing
Ulrich
U
Czibere
Akos
A
Prall
Wolf C
WC
Porter
Chris
C
Ramoni
Marco F
MF
Libermann
Towia A
TA
D007834:Lasers; D040081:Protein Array Analysis / Q000379:methods*; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*