Identification of members of the annexin family in the detergent-insoluble fraction of rat Morris hepatoma plasma membranes.

For proteomic analysis, plasma membranes of rat hepatocellular carcinoma Morris hepatoma 7777 were selectively solubilized according to the previously developed method [D. Josic, K. Zeilinger, Methods Enzymol. 271 (1996) 113-134]. If the Triton X100 insoluble pellet is subsequently extracted, several proteins can be solubilized. These proteins can be classified in two groups according to their molecular size. The proteins with apparent molecular weights in SDS-PAGE between 70 and 75 kDa belong to the first group. Smaller proteins, with apparent molecular weights between 30 and 45 kDa, are members of the second group. The main protein of higher molecular weight was also found in the Triton X100 insoluble extract from normal rat liver plasma membranes. This protein was identified as Annexin A6. The proteins from the second group are practically absent in the Triton X100 insoluble extract from rat liver. These proteins are present in relatively high concentrations in plasma membranes of Morris hepatoma 7777. Both groups of detergent-insoluble proteins from Morris hepatoma 7777 were further analyzed with SELDI-TOF and LC electrospray ionization mass spectrometry. From the first group, Annexin A6, together with two other integral plasma membrane proteins, was identified. In the second group of proteins with apparent molecular weights between 30 and 45kDa, further members of the annexin family, Annexins A1, A2, A4, A5 and A7 were identified. The possible role of these low molecular size annexins as potential cancer biomarkers is discussed.

Proteomic characterization of inter-alpha inhibitor proteins from human plasma.

Inter-alpha inhibitor proteins (IaIp) are a family of structurally related serine protease inhibitors found in relatively high concentrations in human plasma. Recent studies have implicated a role for IaIp in sepsis, and have demonstrated their potential as biomarkers in sepsis and cancer. For characterization of isolated IaI proteins and contaminating proteins during the last steps of the purification process, SELDI-TOF MS and HPLC-ESI-MS/MS were used. After separation by SDS-PAGE or 2-DE, polypeptide bands of 80, 125 and 250 kDa were excised from gels and digested by trypsin. The tryptic peptides were analyzed by both MS methods. The main contamination during the purification process, a band of 80 kDa, contains mainly IaIp heavy chain (HC) H3. HC H1 and H2 were also found in this band. In addition, some vitamin K-dependent clotting factors and inhibitors and other plasma proteins were identified. The 125-kDa band, representing the pre-alpha inhibitor, was found to contain both bikunin and HC H3. The presence of other HC H1, H2 and the recently described HC H4 was also detected by SELDI-TOF MS. The presence of HC H1, H2, and H3 in the 125-kDa band was confirmed by ESI-MS/MS, but not the presence of the H4. Three polypeptides, H1 and H2 together with bikunin, were identified in the 250-kDa band, representing the ITI, by both MS techniques. Once again, the presence of H4 was detected in this band only by SELDI-TOF MS, but the number of corresponding peptides was still not sufficient for final identification of this polypeptide. The importance of the application of proteomic methods for the proper evaluation of therapeutic drugs based on human plasma is discussed.

Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins.

A model system for selective solubilization and fast separation of proteins from the rat liver membrane fraction and purified rat liver plasma membranes for their further proteomic analysis is presented. For selective solubilization, high-pH solutions and a concentrated urea solution, combined with different detergents, are used. After extraction, proteins are separated by anion-exchange chromatography or a combination of anion- and cation-exchange chromatography with convective interaction monolithic supports. This separation method enables fast and effective prefractionation of membrane proteins based on their hydrophobicity and charge prior to one-dimensional (1-D) and 2-D electrophoresis and mass spectrometry. By use of this sample preparation method, the less-abundant proteins can be detected and identified.