Ultrasensitive Proteomics Using High-Efficiency On-Line Micro-SPE-NanoLC-NanoESI MS and MS/MS
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- 作者：Yufeng Shen ; Nikola Toli
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- 刊名：Analytical Chemistry
- 出版年：2004
- 出版时间：January 1, 2004
- 年：2004
- 卷：76
- 期：1
- 页码：144 - 154
- 全文大小：460K
- 年卷期：v.76,no.1(January 1, 2004)
- ISSN：1520-6882
文摘
Ultrasensitive nanoscale proteomics approaches for characterizing proteins from complex proteomic samples of<50 ng of total mass are described. Protein identificationsfrom 0.5 pg of whole proteome extracts were enabled byultrahigh sensitivity (<75 zmol for individual proteins)achieved using high-efficiency (peak capacities of ~103)15-
m-i.d. capillary liquid chromatography separations(i.e., using nanoLC, ~20 nL/min mobile-phase flow rateat the optimal linear velocity of ~0.2 cm/s) coupled on-line with a micro-solid-phase sample extraction and ananoscale electrospray ionization interface to a 11.4-TFourier transform ion cyclotron resonance (FTICR) massspectrometer (MS). Proteome measurement coverageimproved as sample size was increased from as little as0.5 pg of sample. It was found that a 2.5-ng sampleprovided 14% coverage of all annotated open readingframes for the microorganism Deinococcus radiodurans, consistent with previous results for a specificculture condition. The estimated detection dynamic rangefor detected proteins was 105-106. An improved accuratemass and LC elution time two-dimensional data analysismethodology, used to both speed and increase the confidence of peptide/protein identifications, enabled identification of 872 proteins/run from a single 3-h nanoLC/FTICR MS analysis. The low-zeptomole-level sensitivityprovides a basis for extending proteomics studies tosmaller cell populations and potentially to a single mammalian cell. Application with ion trap MS/MS instrumentation allowed protein identification from 50 pg (totalmass) of proteomic samples (i.e., ~100 times larger thanFTICR MS), corresponding to a sensitivity of ~7 amol forindividual proteins. Compared with single-stage FTICRmeasurements, ion trap MS/MS provided a much lowerproteome measurement coverage and dynamic range fora given analysis time and sample quantity.
m-i.d. capillary liquid chromatography separations(i.e., using nanoLC, ~20 nL/min mobile-phase flow rateat the optimal linear velocity of ~0.2 cm/s) coupled on-line with a micro-solid-phase sample extraction and ananoscale electrospray ionization interface to a 11.4-TFourier transform ion cyclotron resonance (FTICR) massspectrometer (MS). Proteome measurement coverageimproved as sample size was increased from as little as0.5 pg of sample. It was found that a 2.5-ng sampleprovided 14% coverage of all annotated open readingframes for the microorganism Deinococcus radiodurans, consistent with previous results for a specificculture condition. The estimated detection dynamic rangefor detected proteins was 105-106. An improved accuratemass and LC elution time two-dimensional data analysismethodology, used to both speed and increase the confidence of peptide/protein identifications, enabled identification of 872 proteins/run from a single 3-h nanoLC/FTICR MS analysis. The low-zeptomole-level sensitivityprovides a basis for extending proteomics studies tosmaller cell populations and potentially to a single mammalian cell. Application with ion trap MS/MS instrumentation allowed protein identification from 50 pg (totalmass) of proteomic samples (i.e., ~100 times larger thanFTICR MS), corresponding to a sensitivity of ~7 amol forindividual proteins. Compared with single-stage FTICRmeasurements, ion trap MS/MS provided a much lowerproteome measurement coverage and dynamic range fora given analysis time and sample quantity.