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Stable Isotope Labeling of Amino Acids in Flies (SILAF) Reveals Differential Phosphorylation of Mitochondrial Proteins Upon Loss of OXPHOS Subunits.

ABSTRACT: Drosophila melanogaster has been a workhorse of genetics and cell biology for more than a century. However, proteomic-based methods have been limited due to the complexity and dynamic range of the fly proteome and the lack of efficient labeling methods. Here, we advanced a chemically defined food source into direct stable-isotope labeling of amino acids in flies (SILAF). It allows for the rapid and cost-efficient generation of a large number of larvae or flies, with full incorporation of lysine-[¹³C₆] after six labeling days. SILAF followed by fractionation and enrichment gave proteomic insights at a depth of 7196 proteins and 8451 phosphorylation sites, which substantiated metabolic regulation on enzymatic level. We applied SILAF to quantify the mitochondrial phosphoproteome of an early-stage leucine-rich PPR motif-containing protein (LRPPRC)-knockdown fly model of mitochondrial disease that almost exclusively affects protein levels of the oxidative phosphorylation (OXPHOS) system. While the mitochondrial compartment was hypo-phosphorylated, two conserved phosphosites on OXPHOS subunits NDUFB10 and NDUFA4 were significantly upregulated upon impaired OXPHOS function. The ease and versatility of the method actuate the fruit fly as an appealing model in proteomic and posttranslational modification studies, and it enlarges potential metabolic applications based on heavy amino acid diets.

SUBMITTER: Schober FA

PROVIDER: S-EPMC8050774 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Signals that control responses to stimuli and cellular function are transmitted through the dynamic phosphorylation of thousands of proteins by protein kinases. Many techniques have been developed to study phosphorylation dynamics, including several mass spectrometry (MS)-based methods. Over the past few decades, substantial developments have been made in MS techniques for the large-scale identification of proteins and their post-translational modifications. Nevertheless, all of the current MS-based techniques for quantifying protein phosphorylation dynamics rely on the measurement of changes in peptide abundance levels, and many methods suffer from low confidence in phosphopeptide identification due to poor fragmentation. Here we have optimized an approach for the stable isotope labeling of amino acids by phosphate using [γ-¹⁸O₄]ATP in nucleo to determine global site-specific phosphorylation rates. The advantages of this metabolic labeling technique are increased confidence in phosphorylated peptide identification, direct labeling of phosphorylation sites, measurement phosphorylation rates, and the identification of actively phosphorylated sites in a cell-like environment. In this study we calculated approximate rate constants for over 1,000 phosphorylation sites based on labeling progress curves. We measured a wide range of phosphorylation rate constants from 0.34 min⁻¹ to 0.001 min⁻¹. Finally, we applied stable isotope labeling of amino acids by phosphate to identify sites that have different phosphorylation kinetics during G1/S and M phase. We found that most sites had very similar phosphorylation rates under both conditions; however, a small subset of sites on proteins involved in the mitotic spindle were more actively phosphorylated during M phase, whereas proteins involved in DNA replication and transcription were more actively phosphorylated during G1/S phase. The data have been deposited to the ProteomeXchange with the identifier PXD000680.

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Stable Isotope Labeling of Amino Acids in Flies (SILAF) Reveals Differential Phosphorylation of Mitochondrial Proteins Upon Loss of OXPHOS Subunits.

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