Ernoult Emilie, Gamelin Erick, Guette Catherine
Centre INSERM Régional de Recherche sur le Cancer U, Angers, France.
Proteome Sci. 2008 Oct 13;6:27. doi: 10.1186/1477-5956-6-27.
The development of mass spectrometric techniques and fractionation methods now allows the investigation of very complex protein mixtures ranging from subcellular structures to tissues. Nevertheless, this work is particularly difficult due to the wide dynamic range of protein concentration in eukaryotic tissues. In this paper, we present a shotgun method whereby the peptides are fractionated using OFFGEL electrophoresis after iTRAQ labelling.
We demonstrated that iTRAQ peptide labelling enhances MALDI ionisation and that the OFFGEL fractionation of the labelled peptides introduces a supplementary criterion (pI) useful for validation and identification of proteins. We showed that iTRAQ samples allowed lower-concentrated proteins identification in comparison with free-labelled samples.
The combined use of iTRAQ labelling and OFFGEL fractionation allows a considerable increase in proteome coverage of very complex samples prepared from total cell extracts and supports the low-concentrated protein identification.
质谱技术和分级分离方法的发展，使得从亚细胞结构到组织等非常复杂的蛋白质混合物的研究成为可能。然而，由于真核组织中蛋白质浓度的动态范围很宽，这项工作极具挑战性。在本文中，我们提出了一种鸟枪法，即在iTRAQ标记后使用等压聚焦电泳对肽段进行分级分离。
我们证明，iTRAQ肽段标记增强了基质辅助激光解吸电离（MALDI），并且标记肽段的等压聚焦分级分离引入了一个有助于蛋白质验证和鉴定的补充标准（等电点）。我们表明，与游离标记样品相比，iTRAQ样品能够鉴定低浓度蛋白质。
iTRAQ标记和等压聚焦分级分离的联合使用，能够显著提高从总细胞提取物制备的非常复杂样品的蛋白质组覆盖率，并有助于低浓度蛋白质的鉴定。
