TurnoveR: A Skyline External Tool for Analysis of Protein Turnover in Metabolic Labeling Studies

Abstract

In spite of its central role in biology and disease, protein turnover is a largely understudied aspect of most proteomic studies due to the complexity of computational workflows that analyze in vivo turnover rates. To address this need, we developed a new computational tool, TurnoveR, to accurately calculate protein turnover rates from mass spectrometric analysis of metabolic labeling experiments in Skyline, a free and open-source proteomics software platform. TurnoveR is a straightforward graphical interface that enables seamless integration of protein turnover analysis into a traditional proteomics workflow in Skyline, allowing users to take advantage of the advanced and flexible data visualization and curation features built into the software. The computational pipeline of TurnoveR performs critical steps to determine protein turnover rates, including isotopologue demultiplexing, precursor-pool correction, statistical analysis, and generation of data reports and visualizations. This workflow is compatible with many mass spectrometric platforms and recapitulates turnover rates and differential changes in turnover rates between treatment groups calculated in previous studies. We expect that the addition of TurnoveR to the widely used Skyline proteomics software will facilitate wider utilization of protein turnover analysis in highly relevant biological models, including aging, neurodegeneration, and skeletal muscle atrophy.

Numbers of Exchangeable Hydrogens from LC–MS Data of Heavy Water Metabolically Labeled Samples

“…Proteins are continuously synthesized and degraded to maintain the proteome (i.e., proteostasis) appropriate for the cellular environment . Mass spectrometry-based proteomics of metabolically stable-isotope labeled samples is a relatively high-throughput approach to studying turnover rates of individual proteins in vivo . − Among the stable-isotope labeling agents, − deuterated water (D 2 O) offers several advantageous properties such as (1) easy administration of D 2 O, as it can be given orally as drinking water, (2) a low dose of D 2 O is safe for consumption, (3) it is relatively inexpensive, and (4) it freely and rapidly equilibrates with total body water in all organs, organelles, and cells within an organism . For in vivo labeling, the initial bolus of deuterated water (also referred to as heavy water) is followed by the provision of deuterium-enriched (typically around 8%) drinking water for a predetermined period.…”

Flexible Quality Control for Protein Turnover Rates Using d2ome

“…Several software tools [1,8,10,11] have been developed to automate the estimation of protein turnover rate from LC-MS experiments, including d2ome [9,12]. d2ome is a powerful tool for protein turnover estimation from deuterium-labeled LC-MS experiments.…”

Software Tool for Visualization and Validation of Protein Turnover Rates Using Heavy Water Metabolic Labeling and LC-MS

“…A protein turnover estimation software tool [ 12 ] for metabolic labeling with a heavy amino acid ( 13 C 6 -Lys) contained a GUI, ApplE Turnover, to facilitate the data analysis. Another tool for protein turnover estimation from [5,5,5- 2 H 3 ] Lue labeled samples, TurnoveR [ 13 ], used the functions of Skyline [ 14 ], an MS data analysis platform. Here, we report on our implementation of a GUI for a software tool, d2ome [ 10 ], to estimate protein turnover rates from D 2 O labeling.…”