Abstract
Oil bodies, lipid-storage organelles, are stabilized by a number of specific proteins. These proteins are very hydrophobic, which complicates their identification by “classical” proteomic protocols using trypsin digestion. Due to the lack of trypsin cleavage sites, the achievable protein coverage is limited or even insufficient for reliable protein identification. To identify such proteins and to enhance their coverage, we introduced a modified method comprising standard three-step procedure (SDS-PAGE, in-gel digestion, and LC-MS/MS analysis). In this method, chymotrypsin, single or in combination with trypsin, was used, which enabled to obtain proteolytic peptides from the hydrophobic regions and to identify new oil bodies’ proteins. Our method can be easily applied to identification of other hydrophobic proteins.
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Abbreviations
- AA:
-
Acrylamide
- ACN:
-
Acetonitrile
- APS:
-
Ammonium persulfate
- BIS:
-
N,N′-methylenebisacrylamide
- CBB:
-
Coomassie Brilliant Blue
- DTT:
-
Dithiothreitol
- FA:
-
Formic acid
- IAA:
-
Iodoacetamide
- IB:
-
Isolation buffer
- OBs:
-
Oil bodies
- PLB:
-
Protein loading buffer
- SDS:
-
Sodium dodecyl sulfate
- TEMED:
-
N,N,N′,N′-tetramethylethylenediamine
References
Tzen JTC, Huang AHC (1992) Surface structure and properties of plant seed oil bodies. J Cell Biol 117:327–335
Murphy DJ (2001) The biogenesis and functions of lipid bodies in animals, plants and microorganisms. Prog Lipid Res 40:325–438
Poxleitner M, Rogers SW, Samuels AL et al (2006) A role for caleosin in degradation of oil-body storage lipid during seed germination. Plant J 47:917–933
Parthibane V, Rajakumari S, Venkateshwari V et al (2012) Oleosin is bifunctional enzyme that has both monoacylglycerol acyltransferase and phospholipase activities. J Biol Chem 287:1946–1954
Kim HU, Hsieh K, Ratnayake C et al (2002) A novel group of oleosins is present inside the pollen of Arabidopsis. J Biol Chem 277:22677–22684
Vermachova M, Purkrtova Z, Santrucek J et al (2011) New protein isoforms identified within Arabidopsis thaliana seed oil bodies combining chymotrypsin/trypsin digestion and peptide fragmentation analysis. Proteomics 11:3430–3434
Tzen JTC, Peng CC, Cheng DJ et al (1997) A new method for seed oil body purification and examination of oil body integrity following germination. J Biochem 121:762–768
Jolivet P, Roux E, d’Andréa S et al (2004) Protein composition of oil bodies in Arabidopsis thaliana ecotype WS. Plant Physiol Biochem 42:501–509
Neuhoff V, Arold N, Taube D et al (1988) Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9:255–262
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Vermachova, M., Purkrtova, Z., Santrucek, J., Jolivet, P., Chardot, T., Kodicek, M. (2014). Combining Chymotrypsin/Trypsin Digestion to Identify Hydrophobic Proteins from Oil Bodies. In: Jorrin-Novo, J., Komatsu, S., Weckwerth, W., Wienkoop, S. (eds) Plant Proteomics. Methods in Molecular Biology, vol 1072. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-631-3_14
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DOI: https://doi.org/10.1007/978-1-62703-631-3_14
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-630-6
Online ISBN: 978-1-62703-631-3
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