Better Proteomic Measurements Achieved with Smaller Liquid Chromatography Columns
Contact: Keqi Tang
Scientists at Pacific Northwest National Laboratory (PNNL) developed a silica-based monolithic LC column with an inner diameter of 10 micrometers that provides proteomics measurements more than 10 times as sensitive than those of conventional LC. The sensitivity of proteomics measurements using liquid chromatography (LC) separations interfaced with electrospray ionization-mass spectrometry (ESI-MS) improves as the liquid flow rate decreases, making attractive the use of smaller inner-diameter LC columns.
Proteomics analyses—the study of the production and dynamics of proteins in a cell under different conditions—give insight into how biological systems operate. Ultimately PNNL's studies may contribute to solutions to a wide range of human health and environmental problems.
In one example, the researchers identified more than 5000 different peptides by tandem MS from 100 nanograms of a Shewanella oneidensis tryptic digest using an ion trap MS. The low LC flow rates (~10 nanoliters per minute) provide more uniform signal intensities for different peptides and provided improved quantitative measurements compared to conventional separation systems, without the use of internal standards or isotopic labeling. In another example, in conjunction with researchers at Purdue University, the improved sensitivity allowed LC-MS measurements of protein phosphatase 5 (a novel human protein that is a negative modulator of heat shock factor activity) that were also validated using quantitative Western blot analyses. The advantage of the MS-based approach over Western blot analyses is that it is broadly applicable and does not require specific reagents for each protein.
To perform quantitative analyses of proteins in cells or tissues, various combinations of separations with mass spectrometry (MS) are widely applied. The value of these measurements increases with the separations quality, the sensitivity and dynamic range of the MS instrument, and how well the protein abundance aligns with the MS response.
The paper appeared in the May 2006 issue of the Journal of Proteome Research. Authors are Quanzhou Luo, Keqi Tang, Feng Yang, Yufeng Shen, Ron Moore, Rui Zhao, Kim Hixson, and Dick Smith, PNNL; and Ayesha Elias and Sandra Rossie, Purdue University. Portions of this research were supported by the U.S. Department of Energy (DOE) Office of Biological and Environmental Research and the NIH National Center for Research Resources at PNNL.
Diagram of PNNL's microsolid phase extraction-nanoLC system using a 10-micrometer-inner-diameter silica-based monolithic column. At right: peak area versus total amount of sample injected onto the LC column for six high-abundance peptides.
Luo QZ, KQ Tang, F Yang, A Elias, YF Shen, RJ Moore, R Zhao, KK Hixson, SS Rossie, and RD Smith. 2006. "More sensitive and quantitative proteomic measurements using very low flow rate porous silica monolithic LC columns with electrospray ionization-mass spectrometry." J Proteome Res 5(5):1091-1097.