Multidimensional liquid chromatography separation of intact proteins by chromatographic focusing and reversed phase of the human serum proteome: optimization and protein database.

In biomarker discovery, the detection of proteins with low abundance in the serum proteome can be achieved by optimization of protein separation methods as well as selective depletion of the higher abundance proteins such as immunoglobins (e.g. IgG) and albumin. A relative newcomer to the proteomic separation arena is the commercial instrument PF2D from Beckman Coulter that separates proteins in the first dimension using chromatofocusing followed in line by reversed phase chromatography in the second dimension, thereby separating intact proteins based on pI and hydrophobicity. In this study, assessment and optimization of serum separation (undepleted serum and albumin-IgG-depleted serum) by the PF2D is presented. Protein databases were created for serum obtained from a healthy individual under traditional and optimized methods and under different sample preparation protocols. Separation of the doubly depleted serum using the PF2D with 20% isopropanol present in the first dimension running buffer allowed us to unambiguously identify 150 non-redundant serum proteins (excluding all immunoglobulin and albumin, a minimum of two peptide matches with acceptable Mascot score) in which 81 have not been identified previously in serum. Among them, numerous cellular proteins were identified to be specifically the skeletal muscle isoform, such as skeletal muscle fast twitch isoforms of troponin T, myosin alkali light chain 1, and sarcoplasmic/endoplasmic reticulum calcium ATPase. The detection of specific skeletal muscle protein isoforms in the serum from healthy individuals reflects the physiological turnover that occurs in skeletal muscle, which will have an impact on the ability to use generic “cellular” proteins as biomarkers without further characterization of the precise isoforms or post-translational modifications present. Molecular & Cellular Proteomics 5:26–34, 2006. There has been a surge of interest in the proteomic analysis of plasma and serum in the search for clinically relevant biomarkers of disease. In biomarker discovery, it is necessary to maximize the observation of the plasma or serum proteome to detect proteins with low abundance. This can be achieved by optimization of protein separation methods as well as selective depletion of the proteins at high abundance such as immunoglobins (e.g. IgG) and albumin. There are a large number of proteomic technologies that separate either proteins or peptides prior to MS (1). A relative newcomer to the proteomic separation arena is the commercial instrument PF2D from Beckman Coulter that separates proteins in the first dimension using chromatographic focusing followed in line by reversed phase chromatography in the second dimension, thereby separating intact proteins based on pI and hydrophobicity. To date, assessment and optimization of either plasma or serum separation by the PF2D has not been undertaken. Generally LC proteomic methods have focused on separating complex mixtures of peptides obtained following digestion of the serum proteome (peptide LC, shotgun), whereas separation of proteins has been relegated primarily to electrophoresis in both one and two dimensions (2DE) (2, 3). 2DE has an advantage over peptide-based LC methods as it enhances the ability to identify the precise isoforms of proteins that are present and/or post-translational modifications (PTMs) that may alter the pI or mass of a protein (1). The potential to separate proteins, rather than peptides, by liquid chromatography using two dimensions would potentially provide the same advantages as 2DE. The PF2D system (Beckman Coulter) is a two-dimensional LC system that uses chromatographic focusing to separate intact proteins in the first dimension by pI (from 8.5–4.0) and in the second dimension by reversed phase chromatography, which separates based on hydrophobicity, thus enhancing the precise detection of isoforms and/or PTMs that alter the pI and/or hydrophobicity of a protein. Also by resolving proteins based upon their intrinsic characteristics prior to mass spectrometry analysis into fractions containing a few proteins (one up to 20), a high degree of sequence coverage for each identification can be From the Departments of ‡Medicine, §Biological Chemistry, and Biomedical Engineering and ¶The Technical Implementation and Coordination Core of NHLBI Proteomics Center, The Johns Hopkins University, Baltimore, Maryland 21224 Received, August 1, 2005, and in revised form, September 8, 2005 Published, MCP Papers in Press, September 27, 2005, DOI 10.1074/mcp.T500019-MCP200 1 The abbreviations used are: 2DE, two-dimensional electrophoresis; PTM, post-translational modification; TnT, troponin T; sTnT, skeletal troponin T; TnI, troponin I; sTnI, skeletal troponin I; CF, chromatofocusing. Technology