Abstract
Protein turnover is a well-controlled process in which polypeptides are constantly being degraded and subsequently replaced with newly synthesized copies. Extraction of composite spectral envelopes from complex LC/MS shotgun proteomics data can be a challenging task, due to the inherent complexity of biological samples. With partial metabolic labeling experiments this complexity increases as a result of the emergence of additional isotopic peaks. Automated spectral extraction and subsequent protein turnover calculations enable the analysis of gigabytes of data within minutes, a prerequisite for systems biology high throughput studies. Here we present a fully automated method for protein turnover calculations from shotgun proteomics data. The approach enables the analysis of complex shotgun LC/MS 15N partial metabolic labeling experiments. Spectral envelopes of 1419 peptides can be extracted within an hour. The method quantifies turnover by calculating the Relative Isotope Abundance (RIA), which is defined as the ratio between the intensity sum of all heavy (15N) to the intensity sum of all light (14N) and heavy peaks. To facilitate this process, we have developed a computer program based on our method, which is freely available to download at http://promex.pph.univie.ac.at/protover.
Highlights
In shotgun proteomics the FCP (Fold Change in Protein) is widely used to compare protein levels of various samples, but neither resolves the dynamics of the proteome in the different biological states that are being compared, nor the mechanisms whereby the system changes from one state to the other [1,2,3]
The main objective of this work was to develop an efficient algorithm for fully automated protein turnover calculations, which can be applied to any kind of sample data arising from partial metabolic 15N labeling experiments, no matter the type of organism or tissue
Since we cannot assume that every protein will be present in the sample at any given time, the question remains for which proteins/peptides to look in a partial metabolic labeling Liquid Chromatography (LC)/MS shotgun proteomics data set, if this data cannot be used for peptide identification
Summary
In shotgun proteomics the FCP (Fold Change in Protein) is widely used to compare protein levels of various samples, but neither resolves the dynamics of the proteome in the different biological states that are being compared, nor the mechanisms whereby the system changes from one state to the other [1,2,3]. A userfriendly, fully automated, and freely available tool is needed, enabling the extraction of complex partial metabolic labeling data for high throughput studies. The resulting mass spectra of individual proteolytic peptides are a composite of all peptide species of variable 15N incorporation (see example Figure 1) This adds to the inherent complexity of biological shotgunproteomics samples, due to the increased isotopic envelope of individual spectra. The main objective of this work was to develop an efficient algorithm for fully automated protein turnover calculations, which can be applied to any kind of sample data arising from partial metabolic 15N labeling experiments, no matter the type of organism or tissue
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