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 complement and extension to the WormBook chapter Biochemistry and molecular biology, which describes tandem affinity purification (TAP) of protein complexes for analysis by mass spectrometry.
Highlights
C. elegans is an excellent model system for basic biology and increasingly for the study of disease phenotypes (WormBook chapter Obesity and the regulation of fat metabolism; Rodriguez et al, 2013)
For worm CoIP methods, we point the reader to a number of excellent protocols on this topic (Biochemistry and molecular biology; Polanowska et al, 2004; Jedamzik and Eckmann, 2009), to Zanin et al (2011), which provides MS-compatible elution conditions
Protein abundances for comparison between samples are estimated using peptide spectral counts (Liu et al, 2004)—the number of times spectra are identified for a protein, similar to transcript microarray abundance values (Pavelka et al, 2008) or mRNA reads in RNA-Seq—and can be normalized to protein length by a normalized spectral abundance factor (NSAF) (Zybailov et al, 2006)
Summary
C. elegans is an excellent model system for basic biology and increasingly for the study of disease phenotypes (WormBook chapter Obesity and the regulation of fat metabolism; Rodriguez et al, 2013). With the advent of numerous technologies, including mass spectrometry-based proteomics, studies of the protein-protein interactions within C. elegans are becoming commonplace (Tewari et al, 2004; Audhya and Desai, 2008; Biochemistry and molecular biology). These studies are beneficial since they can leverage the wide array of existing information and resources of the C. elegans community to quickly follow-up and understand protein-based discoveries. Throughout this chapter we will describe the methods that can be used to identify post-translational modifications, protein interactions, and complexes, enabling the researcher to exploit the advantages of C. elegans as a model organism to study basic biology and disease mechanisms
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