Abstract
Silkworm (Bombyx mori) is a species of agricultural importance, as well as a model organism for Lepidoptera insects. Proteomic method has been widely used in silkworm research, and a robust mass spectrometry-compatible protein extraction method is urgently needed. In this study, we adapted phenol extraction method to extract silkworm midgut protein, and coupled this method with pH 5 - 8 gel strip for two dimensional electrophoresis. The phenol extraction method significantly increased the resolution, as well as greatly reduced the background of two dimensional electrophoresis gels. In addition, this method was well compatible with mass spectrometry analysis. This is the first report that phenol extraction method is used for silkworm midgut protein extraction, and may be applied in other researches.
Highlights
Proteomic method has been widely used in silkworm research, and a robust mass spectrometry-compatible protein extraction method is urgently needed
Two dimensional electrophoresis (2 - DE) and mass spectrometry methods are commonly used in proteomic studies of many organisms
The authors identified 13 representative proteins on 2 - DE gels by matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry/mass spectrometry (MALDI TOF/TOF MS/MS), suggesting the Borax/ PVPP/Phenol method was compatible with MS [3]
Summary
Two dimensional electrophoresis (2 - DE) and mass spectrometry methods are commonly used in proteomic studies of many organisms. Many factors can influence the quality of 2 - DE map Among those factors, a suitable protein extraction method is the most important one [1]. Wang et al used a modified Borax/PVPP/Phenol method for protein preparation from different latex subcellular fractions, and used the resulting protein to construct high-resolution reference 2 - DE maps [3]. Using this method, the protein product from total latex and Cserum fraction generated 1358 and 1248 protein spots on coomassie brilliant blue stained 2 - DE gels, respectively. To verify phenol extraction method was compatible with MS, we selected protein spots from phenol extraction method for MALDI-TOF MS analysis
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