Cells are expressing thousands of proteins at any time, but disease or biological responses may change the expression and interaction of only a few. Chromatography separates molecules based on their chemical properties. High-pressure liquid chromatographs (HPLC) pushes a high-pressure liquid mobile phase through a column packed tightly with particles of a stationary phase. The relative affinity of a substance between the stationary and mobile phase determines how rapidly it moves through the column, accomplishing separation of mixtures.
HPLC in Expression Profiling
The differences in properties that may be used to separate proteins in their natural state are complex. Researchers in the lab of Matthias Mann at the Max Planck Institute of Biochemistry developed a method to perform a proteome-wide screen using ultra high-pressure liquid chromatography (UHPLC) and mass spectrometry (MS). Using only pipette-based techniques, whole-protein extracts were prepared for separation by reverse-phase chromatography, using a polar mobile phase and a nonpolar stationary phase. UHPLC was performed on a Easy-nLC 1000 (Thermo Scientific) and peptides were identified using a Q Exactive Mass Spectrometer (Thermo Scientific). Full analysis of a sample took 4 hours.1,2
Reliable Expression Profiles in a Single Run
A single-shot run detected an average of 3,923 proteins of the approximately 4,000 yeast express under laboratory conditions. A limitation of shotgun proteomic screens is the missing value problem where proteins may be detected in one trail, but not another. In six of six trials, 92% of detected proteins were present. The expression level of proteins was inferred from, on average, seven peptides identified using this LC-MS/MS method covering a median of 23% of the overall protein sequence. This analysis was highly sensitive to changes in the proteome during the yeast heat-shock response.1
Expression profiling using tandem chromatography and mass spectrometry typically requires specialized expertise to execute. Mann’s method expands the utility of LC-MS/MS profiling to an increasing number of labs utilizing proteomics to research the basic science of cellular physiology and the changes underlying disease.
1. Nagaraj, N., et al. (2011) ‘System-wide perturbation analysis with nearly complete coverage of the yeast proteome by single-shot ultra HPLC runs on a bench top Orbitrap‘, Molecular and Cellular Proteomics, 11 (3), published online October 20, 2011. doi: 10.1074/mcp.M111.013722
2. Thakur, S.S., et al. (2011) ‘Deep and highly sensitive proteome coverage by LC-MS/MS without prefractionation‘, Molecular and Cellular Proteomics, 10 (8), published online May 17, 2011. doi: 10.1074/mcp.M110.003699