d2ome, Software for in Vivo Protein Turnover Analysis Using Heavy Water Labeling and LC–MS, Reveals Alterations of Hepatic Proteome Dynamics in a Mouse Model of NAFLD

Abstract

Metabolic labeling with heavy water followed by LC-MS is a high throughput approach to study proteostasis in vivo. Advances in mass spectrometry and sample processing have allowed consistent detection of thousands of proteins at multiple time points. However, freely available automated bioinformatics tools to analyze and extract protein decay rate constants are lacking. Here, we describe d2ome—a robust, automated software solution for in vivo protein turnover analysis. d2ome is highly scalable, uses innovative approaches to nonlinear fitting, implements Grubbs’ outlier detection and removal, uses weighted-averaging of replicates, applies a data dependent elution time windowing, and uses mass accuracy in peak detection. Here, we discuss the application of d2ome in a comparative study of protein turnover in the livers of normal vs Western diet-fed LDLR–/– mice (mouse model of nonalcoholic fatty liver disease), which contained 256 LC-MS experiments. The study revealed reduced stability of 40S ribosomal protein subunits in the Western diet-fed mice.

Early Pro-Inflammatory Remodeling of HDL Proteome in a Model of Diet-Induced Obesity: 2H2O-Metabolic Labeling-Based Kinetic Approach

“…The rate of labeled amino acids incorporation into the specific protein depends on the rate of synthesis of the protein. Tryptic peptides are analyzed by high-resolution mass spectrometry and protein turnover rates are assessed based on 2 H enrichment of peptides ( Figure 1) [22]. We have previously applied this method to a mouse model of NAFLD and demonstrated that HFD results in an increased degradation of plasma and hepatic mitochondrial proteins [23][24][25].…”

“…3 of 20 [22]. We have previously applied this method to a mouse model of NAFLD and demonstrated that HFD results in an increased degradation of plasma and hepatic mitochondrial proteins [23][24][25].…”

“…The data were searched using Mascot software (Matrix Science, London, UK) version 2.3 against the mouse subset of the SwissProt protein database released in October 2016 (containing 552,884 entries) with cysteine carbamidomethylation as a fixed modification and methionine oxidation as a variable modification and trypsin digestion with a maximum of two missed cleavages per peptide. 2 H-labeling of the peptides precursor ions from high-resolution full scan (MS1) spectra was extracted for each isotopomer [22]. Isotopomers are molecules that differ by the presence of isotopes in different positions resulting in an isotopomer pattern in a mass spectrum.…”

Temporal Dynamics of High-Density Lipoprotein Proteome in Diet-Controlled Subjects with Type 2 Diabetes

“…A custom-built software was used for protein, peptide listing, and to calculate isotopic enrichment (E) of the 2 H atoms incorporated into tryptic peptides at each time-point [38]. The proteins identified by the Mascot search engine were filtered based on distinct peptides, peptide and protein score thresholds, and at least four experiments in which a peptide/protein was consistently identified.…”

High-Resolution Mass Spectrometry for In Vivo Proteome Dynamics using Heavy Water Metabolic Labeling

“…At low doses of exposure, no adverse effects have been observed in humans who have continuously consumed 2 H 2 O-enriched water for several months [ 5 , 6 ]. Using the time course of deuterium incorporation, the proteome dynamics of various tissues such as liver [ 4 , 7 , 8 ], heart [ 3 , 9 ], kidney [ 9 ], and muscle [ 10 , 11 ], as well as blood plasma [ 12 , 13 ], have been studied.…”