2-Dimensional gel electrophoresis technique for yeast selenium-containing proteins ― sample preparation and MS approaches for processing 2-D gel protein spots

摘要

A novel integrated approach is proposed for the analysis of intact yeast selenium-containing proteins purified by a gel electrophoresis technique. The strategy consists of three components: high-resolution two-dimensional . gel electrophoresis (2-DE) for proteins, laser ablation-inductively coupled plasma-dynamic reaction cell-mass spectrometry (LA-ICP-DRC-MS) for selenium detection and protein characterisation by electrospray mass spectrometry (ion-trap (IT MS) and time-of-flight (TOF MS) instruments). High-resolution 2-DE is the method of choice for protein purification before their characterisation by mass spectrometry techniques. SDS-PAGE which is a denaturating technique is applicable in this case as the selenium is covalently bound to the proteins. LA-ICP-DRC-MS was used, as optimised in previous work for several elements, which also showed that the limit of detection (absolute LOD of 0.5 pg Se per crater or 70 ng Se g~(-1) gel) was low enough to detect Se in the protein spots. Protein spots were excised from 2-D gels, destained and extracted. Information on the mass of Se-containing proteins were obtained from IT MS and TOF MS. Measurements of the proteins were made with two different MS instruments in order to validate the results and obtain the highest accuracy and resolution on the molecular masses. Up to ten selenium-containing proteins were characterised in terms of molecular mass in the range 9-20 kDa. This strategy has particular application to the possible establishment of a 2-D reference map (Se content and molecular masses) for Se-containing proteins in yeast.