Mass spectrometry-based shotgun proteomic analysis of C. elegans protein complexes

Abstract

Mass spectrometry (MS)-based shotgun proteomics is an enabling technology for the study of C. elegans proteins. When coupled with co-immunoprecipitation (CoIP), new interactions and functions among proteins can be discovered. We provide a general background on protein complexes and methods for their analysis, along with the lifecycle and interaction types of proteins that ultimately define the identifiable components of protein complexes. We highlight traditional biochemical methods to evaluate whether the complexes are sufficiently pure and abundant for analysis with shotgun proteomics. We present two CoIP-MS case studies of protein complexes from C. elegans, using both endogenous and fusion protein antibodies to illustrate the important aspects of their analyses. We discuss results from mass spectrometers with differences in mass accuracy and resolution, along with the relevant information that can be extracted from the data generated, such as protein relative abundance, post-translational modifications, and identification confidence. Finally, we illustrate how comparative analysis can reveal candidate binding partners for biological follow-up and validation. This chapter should act as a compliment and extension to Walhout and Boulton’s 2006 Molecular Biology and Biochemistry Chapter (Walhout and Boulton, 2006) which describes tandem affinity purification (TAP) of protein complexes for analysis by mass spectrometry.

Quantitative multiplexed proteomics of Taenia solium cysts obtained from the skeletal muscle and central nervous system of pigs

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Characterization of TAG-63 and its role on axonal transport inC. elegans

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CBD-1 scaffolds two independent complexes required for eggshell vitelline layer formation and egg activation in C. elegans

“…Crude extract was prepared from POM24 (PERM-4::mCherry; GFP::PH) and POM27 (PERM-2::mCherry; GFP::PH) adult worms by sonication in 10 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM EDTA and 0.5% NP-40, and the soluble fraction was incubated with RFP-Trap magnetic agarose beads as recommended by the manufacturer (Chromotek). Immunoprecipitated proteins were eluted in 50 mM Tris pH8.5 containing 8M urea (Cheeseman et al, 2004) and processed for protein identification by mass spectrometry (Fonslow, 2014).…”