Proteome profile of human urine with two‐dimensional liquid phase fractionation

Abstract

Two-dimensional liquid chromatography separation (2-DL), based on chromatofocusing for first dimension and hydrophobicity for second, can be used as a complementary method to two-dimensional gel electrophoresis (2-DE). A platform now available, ProteomeLab PF 2D provided by Beckman Coulter, (Fullerton, CA, USA), assembles these methods in automation. This system was applied to resolve large numbers of urine proteins. Reproducibility and sensitivity in protein resolution were evaluated in this study using urines collected from male blood donors. About 1000 peaks were detected at a pH range of 4.0-8.5 by applying 1 mg of proteins. Furthermore, the same fractions showing peaks with high absorbance intensities in second dimension were collected and subjected to matrix-assisted laser desorption/ionization-time of flight/mass spectrometry analysis for identification. The results showed that the 2-DL provides high reproducibility of two-dimensional protein map, and lends fractions to subsequent mass spectrometry analysis without the further need for extraction or solubilization of samples as required for spots excised from 2-DE gels. In addition, this system also allows to separate particularly proteins with 40-9 kDa molecular weight.

Advances in Urinary Proteome Analysis and Biomarker Discovery

“…A sequential separation using different media in two independent steps provides a multidimensional fractionation that can generate vast amounts of information. For example, the multidimensional protein identification technology (MudPIT) (27,28) and a 2D liquid-phase fractionation approach (29) are well suited for an in-depth analysis of body fluids and tissues ( Figure 3). Limitations of LC-MS include difficulties with comparative analysis, in part because of the variability in multidimensional separations and the substantial time required for the analysis of a single sample (generally, in days).…”

Rapid T cell–based identification of human tumor tissue antigens by automated two-dimensional protein fractionation

“…immunogenic epitope in more detail, we synthesized overlapping 20-meric peptides of the respective region ( Figure 6C) and identified S100A9 [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] as the immunogenic epitope ( Figure 6D). This epitope was recognized both by CD8 + and CD4 + T cells of patient NCH550 ( Figure 6E).…”

“…This novel method - 2-dimensional (2D) chromatography - is a potent tool for separating proteins from tumor proteomes and represents an alternative to 2D gel electrophoresis (10). Fractioning by using a PF2D system is based on the isoelectric point (pI) and hydrophobicity of processed proteins, and the procedure is highly reproducible (10,11). The resulting separated, matrixfree proteins are extracted as fluids and therefore are immediately accessible for further functional analysis, for example, for natural processing by antigen-presenting cells such as DCs.…”

High‐resolution proteome/peptidome analysis of peptides and low‐molecular‐weight proteins in urine

“…A sequential separation using different media in two independent steps provides a multidimensional fractionation that can generate vast amounts of information. Multidimensional protein identification technology (MudPIT) [25,26,27,28] or a 2D liquid-phase fractionation approach [29] is well suited for in-depth analysis of body fluids.…”