Abstract
BackgroundQuantitative measurements of specific protein phosphorylation sites, as presented here, can be used to investigate signal transduction pathways, which is an important aspect of cell dynamics. The presented method quantitatively compares peptide abundances from experiments using 18O/16O labeling starting from elaborated MS spectra. It was originally developed to study signaling cascades activated by amyloid-β treatment of neurons used as a cellular model system with relevance to Alzheimer's disease, but is generally applicable.ResultsThe presented method assesses, in complete cell lysates, the degree of phosphorylation of specific peptide residues from MS spectra using 18O/16O labeling. The abundance of each observed phospho-peptide from two cell states was estimated from three overlapping isotope contours. The influence of peptide-specific labeling efficiency was removed by performing a label swapped experiment and assuming that the labeling efficiency was unchanged upon label swapping. Different degrees of phosphorylation were reported using the fold change measure which was extended with a confidence interval found to reflect the quality of the underlying spectra. Furthermore a new way of method assessment using simulated data is presented. Using simulated data generated in a manner mimicking real data it was possible to show the method's robustness both with increasing noise levels and with decreasing labeling efficiency.ConclusionThe fold change error assessable on simulated data was on average 0.16 (median 0.10) with an error-to-signal ratio and labeling efficiency distributions similar to the ones found in the experimentally observed spectra. Applied to experimentally observed spectra a very good match was found to the model (<10% error for 85% of spectra) with a high degree of robustness, as assessed by data removal. This new method can thus be used for quantitative signal cascade analysis of total cell extracts in a high throughput mode.
Highlights
Quantitative measurements of specific protein phosphorylation sites, as presented here, can be used to investigate signal transduction pathways, which is an important aspect of cell dynamics
The samples were subsequently analyzed by mass spectrometry and the acquired spectra were initially processed through a series of analysis steps, which are not part of the method presented and not detailed here
To measure the quality of the estimated fold change we introduce the fold change error defined below as the difference between true fold change and the fold change estimated from the model based on the spectra: fold change error =| true fold change − estimated fold change |
Summary
Quantitative measurements of specific protein phosphorylation sites, as presented here, can be used to investigate signal transduction pathways, which is an important aspect of cell dynamics. The experimental setup uses stable isotope labeling by normal or heavy oxygen (16O or 18O) to differentiate between mixed treated and control peptides[2] This peptide mixture is analyzed by mass spectrometry in a single run. The proteins were extracted from the treated and untreated cells, an aliquot split was performed followed by 18O/16O C-terminal labeling by trypsination in two independent experiments (see Methods). This produced a 'direct' experiment, where the peptides from the treated cells were labeled with heavy oxygen (18O) and mixed with peptides from the untreated control cells labeled with light oxygen (16O), and an 'inverted' experiment where the labeling was swapped. The samples were subsequently analyzed by mass spectrometry and the acquired spectra were initially processed through a series of analysis steps (see Methods), which are not part of the method presented and not detailed here
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