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PubMed · 16552026

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·Tesshi Yamada
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Abstract

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-microg 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 10(3)) of our platform warrant its application to various types of experimental and translational proteomics.

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ActininAutomationBlood CellsCell MovementChromatography, High Pressure LiquidColorectal NeoplasmsDiagnostic ImagingDoxycyclineGene ExpressionGenes, ReporterHumansMass SpectrometryMicrofilament ProteinsMicrofluidic Analytical TechniquesPancreatic NeoplasmsPeptide FragmentsProteomeProteomicsSoftwareTransfectionTumor Cells, CulturedComparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tValidation Study

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Masaya Ono, Miki Shitashige, Kazufumi Honda, Tomohiro Isobe, Hideya Kuwabara, Hirotaka Matsuzuki, Setsuo Hirohashi, Tesshi Yamada. 2006-03-21. Label-free quantitative proteomics using large peptide data sets generated by nanoflow liquid chromatography and mass spectrometry.. https://doi.org/10.1074/mcp.t500039-mcp200

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