Combined 2D electrophoretic approaches for the study of white lupin mature seed storage proteome

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• 作者：Chiara Magni ; Alessio Scarafoni ; Anita Herndl ; Fabio Sessa ; Bhakti Prinsi ; Luca Espen ; Marcello Duranti
• 关键词：Liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) ; Lupinus albus ; Legume seed ; Proteome analysis ; Seed storage proteins ; 2D Electrophoresis
• 刊名：Phytochemistry
• 出版年：2007
• 期刊代码：85_00319422
• 类别：ch
• 出版时间：April 2007
• 卷：68
• 期：7
• 页码：997-1007
• 文件大小：2007 K

摘要

Seed proteome analysis by 2D IEF/SDS–PAGE techniques is challenging for the intrinsic difficulties related to quantitative disparity of the seed proteins, i.e. storage and non-storage proteins, their polymorphic nature, the extensive post-translational modifications and the paucity of deposited primary structures available. Conversely, 2D maps of seed proteomes can be extremely useful for a number of fundamental and applied investigations.

In this work, we have used a combination of two experimental approaches to identify the main protein components of an emerging protein-rich legume seed, that is white lupin seed (Lupinus albus, L.). One is the canonical proteomic approach including 2D electrophoretic separation and mass spectrometry of selected trypsin-digested polypeptides; the other approach is a group comparative 2D electrophoretic analysis of cotyledonary protein families. To this second purpose, the three main families of lupin seed proteins, namely α-conglutins, the 11S globulin fraction, β-conglutins, the 7S globulin fraction, and γ-conglutin, a basic 7S protein, were isolated by conventional biochemical techniques and their 2D reference maps were compared with the total protein map.

With the first approach 37 out of 40 spots, making up about 35%of total spot volumes in the 2D map, were found to belong to the main seed protein families. Thanks to cDNA-deduced lupin storage protein sequences, determined on purpose and deposited, most of the identification statistical parameters were very good. Moreover, it was possible to identify several endogenously proteolysed subunits in the map. The second comparative approach, beside confirming these attributions, allowed to allocate 124 polypeptides within the three main lupin protein families.

These two approaches proved to be mutually validating and their combined use was effective for the establishment of a seed proteome map even in the case of sequence and protein post-translational processing lack of information. The results obtained also extend our knowledge of the seed storage protein polymorphism of white lupin.