Abstract
Protein-focused research has been challenging in Drosophila melanogaster due to few specific antibodies for Western blotting and the lack of effective labeling methods for quantitative proteomics. Herein, we describe the preparation of a holidic medium that allows stable-isotope labeling of amino acids in fruit flies (SILAF). Furthermore, in this chapter, we provide a protocol for mitochondrial enrichments from Drosophila larvae and flies together with a procedure to generate high-quality peptides for further analysis by mass spectrometry. Samples obtained following this protocol can be used for various functional studies such as comprehensive proteome profiling or quantitative analysis of posttranslational modifications upon enrichment. SILAF is based on standard fly routines in a basic wet lab environment and provides a flexible and cost-effective tool for quantitative protein expression analysis.
Highlights
Stable-isotope labeling of amino acids in cell culture (SILAC) has been a powerful method to quantitatively assess protein content by high-resolution mass spectrometry (MS)
Cells are grown in a medium that contains one or more non-radioactive isotopes of amino acids [1], typically leucine, lysine, and/or arginine
Upon incorporation of a heavy amino acid isotope during translation, a protein gains discrete mass and its peptides can be distinguished from light isotopic counterparts during an MS analysis
Summary
Stable-isotope labeling of amino acids in cell culture (SILAC) has been a powerful method to quantitatively assess protein content by high-resolution mass spectrometry (MS). Upon incorporation of a heavy amino acid isotope during translation, a protein gains discrete mass and its peptides can be distinguished from light isotopic counterparts during an MS analysis This allows the measurement of two or more differently labeled samples at the same time. Protein quantification in single-shot experiments can be performed cheaper and easier by label-free quantification, a broad range of experiments are typically performed with or require SILAC, among Those deep quantitative proteome profiling using fractionation, differential posttranslational modification quantification, immunoprecipitation, and turnover studies [2]. For Drosophila melanogaster, the fruit fly, a SILAC method was proposed in 2010 [5] and subsequently developed further [6, 7] It relied on labeling of yeast cells with a lysine isotope, which is fed to flies. This technique allows deep insights into the mitochondrial fly proteome across a wide dynamic range, comprising nuclear-encoded proteins and mitochondrial-encoded subunits of the oxidative phosphorylation system
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