Multifactorial Screening Design and Analysis of SELDI-TOF ProteinChip ^® Array Optimization Experiments

Abstract

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry is a powerful tool for rapidly generating protein expression data (peptide and protein profiles) from a large number of samples. However, as with any technology, it must be optimized and reproducible for one to have confidence in the results. Using a classical statistical method called the fractional factorial design of experiments, we assessed the effects of 11 different experimental factors. We also developed several metrics that reflect trace quality and reproducibility. These were used to measure the effect of each individual factor, and the interactions between factors, to determine optimal factor settings and thus ultimately produce the best possible traces. Significant improvements to output traces were seen by simultaneously altering several parameters, either in the sample preparation procedure or during the matrix preparation and application procedure. This has led to the implementation of an improved method that gives a better quality, reproducible, and robust output.

Use of surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) to study protein expression in a rat model of cocaine withdrawal

“…The matrix consists of energy absorbing molecules that cocrystallize with protein molecules retained on chip surfaces and facilitates their ionization and desorption (transfer from solid to gaseous phase and acceleration in the vacuum tube; Cordingley et al, 2003;Jock et al, 2004). Ciphergen protocols call for the matrix to be applied to the chip surface soon after the last sample wash.…”

“…Thus, the manner in which matrix (SPA) is applied to the chips may affect the ionization and desorption of protein and peptide molecules and likewise influence the generation of mass spectra (Cordingley et al, 2003;Jock et al, 2004;Vorm et al, 1994). Indeed, the age and application timing of matrix have been reported to influence the quality of spectra observed (Cordingley et al, 2003;Jock et al, 2004;Vorm et al, 1994). To assess the time frame during which the matrix should be applied onto protein chips (application time), matrix was applied at different time points after the last water wash (Fig.…”

Biomarker discovery from body fluids using mass spectrometry

“…Moreover, differences in the spectra depending on the batch of the resin employed have been reported [15,26]. Other variables of the extraction process, such as the concentration and volume of the analyte, the number of washes of the resin, the composition of the washing and elution solutions, the shape of the resin bed (length and size), and others also influence the spectra [14,19,53].…”

“…The matrixes are diverse chemicals that absorb UV light and transfer the energy to the analyte. Their chemical properties, the proportion in which they are mixed with the analyte and the way in which they co-crystallize with the sample influence the spectra and their reproducibility [14,15,19,53].…”

Methodological advances in the discovery of protein and peptide disease markers

“…In general a coefficient of variation (CV) of 8-10% ought to be achievable with this kind of technique but in some studies a much higher CV is noted. This indicates that reproducibility of the surface chemistry may still be an unresolved issue with SELDI and that care needs to be taken to check for reproducibility and repeatability [66]. The lack of direct marker identification is a drawback of present day SELDI-TOF technology.…”