Nanoscale proteomics.
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.
Shen
Y
Y
Biological Science Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
Tolić
N
N
Masselon
C
C
Pasa-Tolić
L
L
Camp
D G
DG
Lipton
M S
MS
Anderson
G A
GA
Smith
R D
RD
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods; D034301:Deinococcus / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D036103:Nanotechnology / Q000379:methods*; D040901:Proteomics / Q000379:methods*
D001426:Bacterial Proteins