Automated Protein Turnover Calculations from ¹⁵N Partial Metabolic Labeling LC/MS Shotgun Proteomics Data

<div><p>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 <a href="http://promex.pph.univie.ac.at/protover" target="_blank">http://promex.pph.univie.ac.at/protover</a>.</p></div

Histograms of the regression coefficient versus the density of proteins.

<p>Histograms of the regression coefficient versus the density of proteins, comparing no filter (A), co-eluted picked peaks filter (B), co-eluted picked peaks and filter noise at TP₀ (C), and all filters combined (D). (A-D) with 422 cases each. For each Time Point, all peptide RIA values (associated with an Accession Number) were averaged. Subsequently the linear regression was calculated, and thereof Histograms produced. (D) includes (the 94 of the 422) proteins that were removed by the post-processing filter.</p

Simulated spectrum of isotopic distribution of the peptide sequence “MPSAVGYQPTLGTEMGTLQER” (charge state 2).

<p>The spectrum consists of a peptide species with natural isotopic distribution (red), a peptide with 30% ¹⁵N incorporation (green), a peptide with 50% ¹⁵N incorporation (blue), and a peptide with 100% ¹⁵N incorporation (purple). The sum of all composite spectra is displayed in black.</p

Histogram plots of the coverage versus frequency of peptides.

<p>The abscissa represents the coverage in percent. The coverage is calculated analogous to formula (III) the sole difference being the constant denominator N_MAX. Frequency of peptides indicates the number of peptide sequences with a given coverage (number of cases 1419). All five Time Points (from A to E, of one biological replicate) from 96 h (A) to 0 h (E), in 24 h intervals of ¹⁵N incorporation are shown.</p

Relative Isotope Abundance (RIA) plots.

<p>The abscissa represents the Time Points (provided by the user in the Experiment File) and the ordinate the RIA ratio at the given Time Point. The titles of the plots indicate the Accession Number for the given data. The legend shows all peptide sequences that could be attributed to the given protein. A: illustrates the RIA plot for G7JAR7 without the application of any filters. B: RIA plot for G7JAR7 with the application of post-processing filter.</p