Franchin Cinzia, Pivato Micaela, Rattazzi Marcello, Arrigoni Giorgio, Millioni Renato
Department of Biomedical Sciences, University of Padova, Via U. Bassi 58/B, 35131 Padova, Italy; Proteomic Center of Padova University, Via G. Orus 2/B, 35129 Padova, Italy.
Proteomic Center of Padova University, Via G. Orus 2/B, 35129 Padova, Italy; Department of Agronomy, Food, Natural Resources, Animals and the Environment, DAFNAE, University of Padova, Viale dell'Università 16, 35020 Legnaro, PD, Italy.
J Chromatogr A. 2014 Aug 15;1355:278-83. doi: 10.1016/j.chroma.2014.05.077. Epub 2014 Jun 5.
Shotgun proteomics of complex samples is generally coupled with at least one peptide fractionation step and, to this effect, peptide isoelectric focusing (IEF) in immobilized pH gradient (IPG) is one of the most used techniques. Fractionation with the OFFGEL 3100 Agilent Technologies apparatus allows the easy recovery of peptides that, after focusing, diffuse into the liquid phase above the gel strip. In this work we investigate the efficiency of peptide diffusion during OFFGEL fractionation and demonstrate that a recovery based only on the spontaneous diffusion process is far from being optimal. We show that a simple additional extraction step with acetonitrile increases of about 40% the amount of material that can be recovered after the focusing. Moreover, we show that the two populations of peptides obtained from the passive elution and from the extraction process are also qualitatively different and only partially overlapping.
复杂样品的鸟枪法蛋白质组学通常与至少一个肽段分级分离步骤相结合，为此，固定化pH梯度（IPG）中的肽段等电聚焦（IEF）是最常用的技术之一。使用安捷伦科技公司的OFFGEL 3100仪器进行分级分离，可轻松回收聚焦后扩散到凝胶条上方液相中的肽段。在这项工作中，我们研究了OFFGEL分级分离过程中肽段扩散的效率，并证明仅基于自发扩散过程的回收率远非最佳。我们表明，使用乙腈进行简单的额外提取步骤，可使聚焦后回收的物质量增加约40%。此外，我们还表明，从被动洗脱和提取过程中获得的两类肽段在质量上也有所不同，且只是部分重叠。
