Thiophilic adsorption revisited.

Specific and efficient selection of serum immunoglobulins, but not other proteins, on T-gel remains difficult. T-gel capacity was determined for different activation conditions and serum loadings. Mass spectrometry analysis was used to identify the proteins found in the flow-through and in the eluted fractions. Alpha-2-macroglobulin and albumin were the major contaminants of the eluates. The influence of the competition between immunoglobulins and the other serum proteins on the adsorption was also studied. Using a serum depleted in immunoglobulins (flow-through of a first chromatography on T-gel), many serum proteins were retained on the T-gel, including albumin. We conclude that T-gel selectivity is less than absolute and may reflect for a large part the experimental conditions of the adsorption.

An efficient proteomics-based approach for the screening of autoantibodies.

This study presents an improved method for the complete transfer of proteins separated by two-dimensional gel electrophoresis to a membrane, specifically designed for the screening and identification of antigens recognized by autoantibodies in patients with breast cancer (BCP) and healthy volunteers. This paper reports the evaluation of this technique using proteins from MCF7 as a source of antigens following 2-DE separation. The appropriate quantity of protein to be loaded on gels (150 microg) has been determined, the aim being a complete and reproducible recovery of all separated proteins onto the polyvinylidene fluoride membrane (2D-blot) after a semi-dry electrotransfer. Several different transfer methods were tested in parallel, resulting in the selection and optimisation of one using a discontinuous buffer system, based on the isotachophoresis theory. To facilitate the comparative analysis of the different sets of 2D-blots probed with individual sera from BCP and healthy volunteers, the 2D-blots were stained with colloidal gold following the immunodetection step. The gels and 2D-blots were scanned and analysed by imaging software. The matching permitted exact localisation of particular relevant protein spots hybridised by antibodies on the 2D-blots. These spots were subsequently located on preparative gels for identification by mass spectrometry. A set of 40 2D-blots was probed with 20 sera from patients with breast cancer and 20 sera from healthy volunteers. In the protein profiles submitted to immunodetection, 15 proteins were repeatedly immunodetected by both BCP and sera from healthy people. Those proteins were identified by mass spectrometry. Conversely, some protein isoforms were preferentially immunodetected by BCP sera and may reflect the presence of this cancer. The improved isotachophoretic method described in this study is suitable for comparing the overall profile of autoimmunity between different populations and for subsequent identification of relevant antigens.

Mitochondrial proteomic approach reveals galectin-7 as a novel BCL-2 binding protein in human cells.

Although the anti-apoptotic activity of Bcl-2 has been extensively studied, its mode of action remains incompletely understood. Deciphering the network of Bcl-2 interacting factors is necessary to better understand the key function of Bcl-2 in apoptosis initiation. To identify novel Bcl-2 mitochondrial partners, we have combined a Bcl-2 immunocapture with a mass spectrometry analysis using highly pure mitochondrial fractions isolated from human cancer cells. We identified at high confidence 127 potential Bcl-2-interacting proteins. Gene ontology mining reveals enrichment for mitochondrial proteins, endoplasmic reticulum-associated proteins, and cytoskeleton-associated proteins. Importantly, we report the identification of galectin-7 (Gal7), a member of a family of ß-galactoside-binding lectins that was already known to exhibit a pro-apoptotic function, as a new mitochondrial Bcl-2 interacting partner. Our data further show that endogenous Bcl-2 coimmunoprecipitates with Gal7 and that recombinant Gal7 directly interacts with recombinant Bcl-2. A fraction of Gal7 is constitutively localized at mitochondria in a Bcl-2-dependent manner and sensitizes the mitochondria to the apoptotic signal. In addition, we show that the Bcl-2/Gal7 interaction is abolished following genotoxic stress. Taken together, our findings suggest that the binding of Gal7 to Bcl-2 may constitute a new target for enhancing the intrinsic apoptosis pathway.

The proteome of the human breast cancer cell line MDA-MB-231: Analysis by LTQ-Orbitrap mass spectrometry.

We have used a combination of SDS-PAGE and LTQ-Orbitrap MS to explore the proteome of the highly invasive MDA-MB-231 breast cancer cell line. Based on about 520 000 MS/MS spectra, a total of 3481 proteins were identified and subsequently classified according to their cellular distribution and molecular function. Interestingly, a large proportion of proteins (38%) were from cellular membranes and we were able to characterize numerous proteins involved in cancer initiation and progression such as the tumor suppressor p53 and the epidermal growth factor receptor. Together, this study represents the largest proteome database of breast cancer cells realized to date and demonstrates the value of using Orbitrap MS for deeper proteome analysis.

Usefulness of autoantigens depletion to detect autoantibody signatures by multiple affinity protein profiling.

Patients with cancer produce specific autoantibodies against protein antigens present in limited amount among a large background of immunoglobulins (Igs), nonrelevant as biomarkers, including natural antibodies. Multiple affinity protein profiling (MAPPing) that combines 2-D immunoaffinity chromatography, enzymatic digestion of the isolated proteins, and identification by MS/MS, may facilitate the identification of these so far unknown patient antibodies. The first immunoaffinity chromatography is crucial, as it is used for selectively removing proteins (autoantigens) recognized by natural antibodies. Application of this depletion step to colon cancer cell proteins is specifically described along with the identification of the natural autoantigens, as well as the coupling of this depletion step with the next steps. By enabling to separate antibody-binding proteins recognized by either natural autoantibodies or patient-specific antibodies this approach may contribute significantly towards the definition of autoantibody signatures.

Proteomic Analysis of the MCF7 Breast Cancer Cell Line.

BACKGROUND: The MCF7 breast cancer cell line is a cellular model for breast cancer studies and marker discovery. Therefore, a better knowledge of its proteome is a prerequisite for a more efficient use of this model. MATERIALS AND METHODS: Proteins expressed during the exponential growth phase of MCF7 cells were analyzed and mapped using two-dimensional gel electrophoresis and mass spectrometry. RESULTS: From the spots excised from preparative gels of whole-cell extracts, a subset of 368 different polypeptides, corresponding to 249 different proteins, was identified. These polypeptides were positioned on a silver-stained gel to construct a reference map. CONCLUSION: The data allowed the construction of the most extensive reference map for MCF7 published to date, with 189 novel proteins, which had not been previously listed on maps, and are now accessible on World 2D-PAGE database, providing a basis for further studies on MCF7.

A proteomic approach to investigate potential biomarkers directed against membrane-associated breast cancer proteins.

The identification of specific protein markers for breast cancer would provide the basis for early diagnosis. Particularly, membrane and membrane-associated proteins are rich in targets for antibodies that may constitute suitable biomarkers of carcinogenesis. However, membrane proteins separation using 2-DE remains difficult. In this work, the breast cancer cell line MCF7 was used as source of proteins for the screening of potential cell membrane-associated antigens recognized by autoantibodies in patients with breast cancer and healthy volunteers. The protein extract obtained using trifluoroethanol (TFE) as cosolvent was compared to a total cell lysate protein extract prepared by a current technique. After 2-DE separation of the two extracts, their protein patterns clearly differed. About 63% of the proteins identified in the TFE-extract were predicted to possess at least one transmembrane domain. 2-D blots probed with sera from cancer patients or from healthy volunteers showed that, as expected, additional antigens were provided in the TFE-extract. Thus, the method described here appeared well suited for proteomic investigation of potential biomarkers undetected by current techniques.

Automating proteome analysis: improvements in throughput, quality and accuracy of protein identification by peptide mass fingerprinting.

The use of robots has major effects on maximizing the proteomic workflow required in an increasing number of high-throughput projects and on increasing the quality of the data. In peptide mass finger printing (PMF), automation of steps downstream of two-dimensional gel electrophoresis is essential. To achieve this goal, the workflow must be fluid. We have developed tools using macros written in Microsoft Excel and Word to complete the automation of our platform. Additionally, because sample preparation is crucial for identification of proteins by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, we optimized a sandwich method usable by any robot for spotting digests on a MALDI target. This procedure enables further efficient automated washing steps directly on the MALDI target. The success rate of PMF identification was evaluated for the automated sandwich method, and for the dried-droplet method implemented on the robot as recommended by the manufacturer. Of the two methods, the sandwich method achieved the highest identification success rate and sequence coverage of proteins.