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.
Highlights
Adaptation of a fully defined, holidic food source for fruit fly SILAC (SILAF) More than 99% labeling efficiency with lysine-6 within one larval generation One milligram of fully lysine-6 labeled peptides for less than $15 14,000 Drosophila phosphorylated sites with 1500 occupancy values Mitochondrial DmLRPPRC1 loss increases phosphorylation of NDUFB10 and NDUFA4
It has led to a reduction in the dependency on techniques such as SILAC, as a quantitative view of the proteome can be obtained by label-free quantification (LFQ)
We find that stable-isotope labeling of amino acids in flies (SILAF) is only marginally more reproducible than LFQ
Summary
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. Stable-isotope labeling of amino acids in cell culture, known as SILAC, uses nonradioactive isotopic labeling to detect differences in relative abundance between at least two peptide populations simultaneously [1] This allows accurate peptide and protein quantification even upon enrichment or fractionation of peptide mixtures and greatly enhances the depth of large-scale screening for proteins, posttranslational modifications [2] or protein–protein interactions [3].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
