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Molecular & Cellular Proteomics 5:1338-1347, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Technology

Label-free Quantitative Proteomics Using Large Peptide Data Sets Generated by Nanoflow Liquid Chromatography and Mass Spectrometry^*

Masaya Ono, Miki Shitashige, Kazufumi Honda, Tomohiro Isobe, Hideya Kuwabara, Hirotaka Matsuzuki, Setsuo Hirohashi and Tesshi Yamada^,¶

From the Chemotherapy Division and Cancer Proteomics Project, National Cancer Center Research Institute, Tokyo 104-0045 and BioBusiness Group, Mitsui Knowledge Industry, Tokyo 164-8555, Japan

We developed an integrated platform consisting of machinery and software modules that can apply vast amounts of data generated by nanoflow LC-MS to differential protein expression analyses. Unlabeled protein samples were completely digested with modified trypsin and separated by low speed (200 nl/min) one-dimensional HPLC. Mass spectra were obtained every 1 s by using the survey mode of a hybrid Q-TOF mass spectrometer and displayed in a two-dimensional plane with m/z values along the x axis, and retention time was displayed along the y axis. The time jitter of nano-LC was adjusted using newly developed software based on a dynamic programming algorithm. The comprehensiveness (60,000–160,000 peaks above the predetermined threshold detectable in 60-µg cell protein samples), reproducibility (average coefficient of variance of 0.35–0.39 and correlation coefficient of over 0.92 between duplicates), and accurate quantification with a wide dynamic range (over 10³) of our platform warrant its application to various types of experimental and translational proteomics.

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