Recent advances have been made in mass spectrometry to achieve high throughput separation and analysis of proteins and peptides with good mass accuracy and resolution. One of the most difficult challenges of the method is the reproducibility of the data over time and between laboratories. Several studies have addressed this issue looking at the impact of preanalytical variables like patient preparation [4], blood sample processing [3], and standardized analytical conditions [8,9]. The main thrust of this study was to examine the effects of spot protocol optimization on spectral quality as determined by number of peaks and signal intensity CVs on a QC sample. There are several laboratory considerations that have previously been reported and were implemented in this study. For example it is widely recommended that sample loading be handled through an automated liquid handling system [6,10], and that they be randomly loaded on the ProteinChips [11]. We used the Biomek 2000 Automation Workstation for these purposes. EAM was applied in two smaller volumes with a constant drying time before the EAM application to increase the number of peaks [6]. Reagent variability was minimized by using reagents from the same manufacturing lot [11] and the possible effects of freezing and thawing and length of time in storage [12] was considered for the serum and fractionated products.
Exp 1 details our first fractionation/profiling methods and Exp 2 our fine-tuning of the initial methodologies. EAM was initially applied manually because of concerns with pipetting such small volumes of highly volatile liquids. However, there were several advantages to automated application, including speed with which this could be performed, and consistent drying times between applications [13]. We also noticed a need to fine-tune spot protocols for each ProteinChip-fraction combination so we defined criteria based on intensity, S/N and resolution of few chosen peaks [8]. This improved the number of peaks detected and the reproducibility of the signal intensities.
By routinely performing and monitoring instrument checks we have established criteria to detect changes in instrument performance [8]. We added a spot-to-spot correction in Exp 2, but found it to have little impact on results.
Comparison on the average peak intensity CVs from Exp 1 to Exp 2 showed a marked improvement, changes of 4% to 37%. H50-F6 and IMAC-F3 did not show improvement. This indicates that the experimental parameters used in Exp 2 provided a considerable improvement in spectral quality. Comparing our results to those of other researchers is complicated due to different experimental conditions – not all sera are fractionated, CVs not calculated for the QC sample alone, different ProteinChips, m/z range differs for data collection, and/or peak selection criteria varies. Koopman et al. [14] used a fractionated QC serum to calculate intra-assay variation on 10 randomly chosen peaks (S/N > 5, m/z [9]. The optimization of the acquisition protocols at the fraction level (Exp 2) and automation of EAM application, have substantially improved the reproducibility of peak intensities.
We used a Kruskal-Wallis non-parametric test, with multiple test correction to examine the variability of peak intensities from the QC sera across the batches in which they were run. Not surprisingly we found that several ProteinChip-fraction combinations had more variability than others. Three ProteinChip-fractions had >30% of their peak intensities being statistically different at p
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