Newly Configured Intact Protein Based Multi Dimensional LC System and Its Application in Complex Proteomics Sample Handling

摘要

>RP-39Comprehensive profiling of the plasma proteomes has substantial relevance to the identification of circulating biomarkers that may have predictive value and utility for disease diagnosis. The substantial complexity and vast dynamic range of protein abundance in biological fluids, notably serum and plasma, present a formidable challenge. It turned out that sample processing was a major problem. Integration of multiple technologies is required to achieve high-resolution and high-sensitivity proteomics sample processing and analysis of biological fluids. We implemented an orthogonal multi-dimensional intact-protein analysis system, coupled with immunodepletion of abundant proteins and protein tagging, to quantitatively profile the human plasma proteome. Samples from carcinomas and matched controls were processed with newly configured Multi Dimmensional LC system. Our results show that the system provides a combination of comprehensive profiling and quantitative analysis, with a substantial dynamic range, for disease-related applications. Applying presented technique enable to keep proteins intact during sample preparation and separation, which has important advantages compared with strategies that digest proteins at an earlier step. For example, post-translational modification (PTM) is believed to be involved in most (nearly all?) protein regulation and signaling, but digestion to peptides was obscuring the signature. Plus, the mass spectrometry was knocking off the weak bounded PTM groups. It was difficult to locate the sites of PTM, and if multiple sites were involved, one had an even harder time studying the distribution of the population of isoforms. The advantages of using the system include the ability to quantify and recover proteins as well as to assess post-translational modifications and cleavage. The data will also show the higher reproducibility, automation and versatility of the system.