ausblenden:
Schlagwörter:
Amino Acids/*chemistry/metabolism
Animals
Culture Media/chemistry
Drosophila melanogaster/*metabolism
Isotope Labeling/*methods
Larva/metabolism
Mass Spectrometry/methods
Mitochondria/metabolism
Peptides/metabolism
Protein Processing, Post-Translational
*Proteome
Proteomics/*methods
*Drosophila
*Proteomics
*SILAC, Mitochondria
*Stable-isotope labeling
Zusammenfassung:
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.
