| A protein profiling method based on chromatofocusing (CF)/non-porous silica reversed phase HPLC (NPS-RP-HPLC) and online ESI TOF has been developed. The application of the method to MCF10A (normal) and Ca1DCL1 (fully malignant) whole cell lysate has revealed new marker proteins, such as 14-3-3 sigma and Histone 2B that are closely related to several signal transduction pathways. The modification of Histone 2B may contribute to the overexpression of CK8 and CK18 in malignant cells. In the CF separation, most proteins elute at their pIs, although differences between the experimental pI and the theoretical pI exist. Such deviations of the experimental pI usually suggests post-translational modifications such as truncation and phosphorylation.; The liquid 2D protein profiling method provides significant improvement on the mass accuracy of intact proteins over 2D gel electrophoresis, where an accuracy of 50ppm for HSP60 (57966Da) is typical. The liquid based method is more compatible to MS analysis since proteins remain soluble during separation. As a result, sample preparation is simplified significantly, which may improve the recovery in sample preparation and sequence coverage. Unlike 2D gel electrophoresis, methionine is free of oxidation in the liquid 2D protein profiling method. Oxidation can be induced when methionine-containing peptides are exposed to light. The location of methionine in the peptide sequence and the presence of modifications do not affect the induced oxidation. Further, proteins with low MW are not biased in liquid 2D separation, where identification can be achieved by using MALDI TOF and MALDI QTOF together with the accurate MW of intact proteins. |
