Proteomic Profiling pp 535-547 | Cite as
Chloroplast Isolation and Enrichment of Low-Abundance Proteins by Affinity Chromatography for Identification in Complex Proteomes
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Abstract
Comprehensive knowledge of the proteome is a crucial prerequisite to understand dynamic changes in biological systems. Particularly low-abundance proteins are of high relevance in these processes as these are often proteins involved in signal transduction and acclimation responses. Although technological advances resulted in a tremendous increase in protein identification sensitivity by mass spectrometry (MS), the dynamic range in protein abundance is still the most limiting problem for the detection of low-abundance proteins in complex proteomes. These proteins will typically escape detection in shotgun MS experiments due to the presence of high-abundance proteins. Therefore, specific enrichment strategies are still required to overcome this technical limitation of MS-based protein discovery. We have searched for novel signal transduction proteins, more specifically kinases and calcium-binding proteins, and here we describe different approaches for enrichment of these low-abundance proteins from isolated chloroplasts from pea and Arabidopsis for subsequent proteomic analysis by MS. These approaches could be extended to include other signal transduction proteins and target different organelles.
Key words
Chloroplast isolation Affinity chromatography Mass spectrometry Proteomics Organelle proteome ATP-binding protein Calcium-binding proteinNotes
Acknowledgments
Work in the authors’ lab is supported by grants from the Austrian Science Fund (FWF) to MT (P 28491-B29) and the FP7 Marie Curie Initial Training Network (ITN) “CALIPSO” (GA ITN 2013-607 607) from the European Union.
References
- 1.Rolland N, Curien G, Finazzi G, Kuntz M, Marechal E, Matringe M et al (2012) The biosynthetic capacities of the plastids and integration between cytoplasmic and chloroplast processes. Ann Rev Genet 46:233–264CrossRefGoogle Scholar
- 2.Jarvis P, Lopez-Juez E (2013) Biogenesis and homeostasis of chloroplasts and other plastids. Nat Rev Mol Cell Biol 14:787–802CrossRefGoogle Scholar
- 3.Kmiecik P, Leonardelli M, Teige M (2016) Novel connections in plant organellar signalling link different stress responses and signalling pathways. J Exp Bot 67:3793–3807CrossRefGoogle Scholar
- 4.Stael S, Kmiecik P, Willems P, Van Der Kelen K, Coll NS, Teige M et al (2015) Plant innate immunity--sunny side up? Trends Plant Sci 20:3–11CrossRefGoogle Scholar
- 5.Zybailov B, Rutschow H, Friso G, Rudella A, Emanuelsson O, Sun Q et al (2008) Sorting signals, N-terminal modifications and abundance of the chloroplast proteome. PLoS One 3:e1994CrossRefGoogle Scholar
- 6.Ferro M, Brugiere S, Salvi D, Seigneurin-Berny D, Court M, Moyet L et al (2010) AT_CHLORO, a comprehensive chloroplast proteome database with subplastidial localization and curated information on envelope proteins. Mol Cell Proteomics 9:1063–1084CrossRefGoogle Scholar
- 7.Huang M, Friso G, Nishimura K, Qu X, Olinares PD, Majeran W et al (2013) Construction of plastid reference proteomes for maize and Arabidopsis and evaluation of their orthologous relationships; the concept of orthoproteomics. J Proteome Res 12:491–504CrossRefGoogle Scholar
- 8.Bouchnak I, Brugiere S, Moyet L, Le Gall S, Salvi D, Kuntz M et al (2019) Unraveling hidden components of the chloroplast envelope proteome: opportunities and limits of better MS sensitivity. Mol Cell Proteomics 18:1285–1306CrossRefGoogle Scholar
- 9.Bayer RG, Stael S, Rocha AG, Mair A, Vothknecht UC, Teige M (2012) Chloroplast-localized protein kinases: a step forward towards a complete inventory. J Exp Bot 63:1713–1723CrossRefGoogle Scholar
- 10.Bayer RG, Stael S, Csaszar E, Teige M (2011) Mining the soluble chloroplast proteome by affinity chromatography. Proteomics 11:1287–1299CrossRefGoogle Scholar
- 11.Kunst L (1998) Preparation of physiologically active chloroplasts from Arabidopsis. Methods Mol Biol 82:43–48PubMedGoogle Scholar
- 12.Brautigam A, Shrestha RP, Whitten D, Wilkerson CG, Carr KM, Froehlich JE et al (2008) Low-coverage massively parallel pyrosequencing of cDNAs enables proteomics in non-model species: comparison of a species-specific database generated by pyrosequencing with databases from related species for proteome analysis of pea chloroplast envelopes. J Biotechnol 136:44–53CrossRefGoogle Scholar
- 13.Kreplak J, Madoui MA, Capal P, Novak P, Labadie K, Aubert G et al (2019) A reference genome for pea provides insight into legume genome evolution. Nat Genet 51:1411–1422CrossRefGoogle Scholar
- 14.Simm S, Papasotiriou DG, Ibrahim M, Leisegang MS, Muller B, Schorge T et al (2013) Defining the core proteome of the chloroplast envelope membranes. Front Plant Sci 4:11CrossRefGoogle Scholar
- 15.Tomizioli M, Lazar C, Brugiere S, Burger T, Salvi D, Gatto L et al (2014) Deciphering thylakoid sub-compartments using a mass spectrometry-based approach. Mol Cell Proteomics 13:2147–2167CrossRefGoogle Scholar
- 16.Wissing J, Jansch L, Nimtz M, Dieterich G, Hornberger R, Keri G et al (2007) Proteomics analysis of protein kinases by target class-selective prefractionation and tandem mass spectrometry. Mol Cell Proteomics 6:537–547CrossRefGoogle Scholar
- 17.Schleiff E, Soll J, Kuchler M, Kuhlbrandt W, Harrer R (2003) Characterization of the translocon of the outer envelope of chloroplasts. J Cell Biol 160:541–551CrossRefGoogle Scholar
- 18.Arnon DI (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta Vulgaris. Plant Physiol 24:1–15CrossRefGoogle Scholar
- 19.Chaga GS, Ersson B, Porath JO (1996) Isolation of calcium-binding proteins on selective adsorbents. Application to purification of bovine calmodulin. J Chromatogr A 732:261–269CrossRefGoogle Scholar
- 20.Halliwell B (1978) The chloroplast at work. A review of modern developments in our understanding of chloroplast metabolism. Prog Biophys Mol Biol 33:1–54CrossRefGoogle Scholar