Nanoscale proteomics.

Abstract

Efforts to develop a liquid chromatography (LC)/mass spectrometry (MS) technology for ultra-sensitive proteomics studies (i.e., nanoscale proteomics) are described. The approach combines high-efficiency nanoscale LC (separation peak capacity of approximately 10(3); 15-microm-i.d. packed capillaries with flow rates of 20 nL min(-1), the optimal separation linear velocity) with advanced MS, including high-sensitivity and high-resolution Fourier transform ion cyclotron resonance MS, to perform both single-stage MS and tandem MS (MS/MS) proteomic analyses. The technology enables broad protein identification from nanogram-size proteomics samples and allows the characterization of more abundant proteins from sub-picogram-size samples. Protein identification in such studies using MS is demonstrated from <75 zeptomole of a protein. The average proteome measurement throughput is approximately 50 proteins h(-1) using MS/MS during separations, presently requiring approximately 3 h sample(-1). Greater throughput (approximately 300 proteins h(-1)) and improved detection limits providing more comprehensive proteome coverage can be obtained by using the "accurate mass and time" tag approach developed in our laboratory. This approach provides a dynamic range of at least 10(6) for protein relative abundances and an improved basis for quantitation. These capabilities lay the foundation for studies from single or limited numbers of cells.