Development of a two-dimensional protein-peptide separation protocol for comprehensive proteome measurements

George M. Janini; Thomas P. Conrads; Timothy D. Veenstra; Haleem J. Issaq

doi:10.1016/S1570-0232(02)00616-5

Development of a two-dimensional protein-peptide separation protocol for comprehensive proteome measurements

George M. Janini, Thomas P. Conrads, Timothy D. Veenstra, Haleem J. Issaq^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

27 Scopus citations

Abstract

We have developed an effective two-dimensional fractionation protocol of complex proteome mixtures that extends the ability to conduct more comprehensive proteome measurements. A sample containing intact proteins extracted from Saccharomyces cerevisiae was fractionated by liquid phase isoelectric focusing, followed by tryptic digestion and solid-phase extraction (SPE) clean-up and reversed-phase liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS-MS) of the resultant peptides. The clean-up step is designed to desalt the fractions and rid them of urea and ampholytes prior to analysis by LC-MS-MS. Fifty milligrams of protein were separated into 20 fractions by liquid-phase isoelectric focusing, spanning a pH range of 3-10. The effectiveness of the removal of ampholytes was monitored by capillary zone electrophoresis and LC-MS-MS. The ability to analyze all of the 20 fractions without any noticeable decrease in the separation efficiency demonstrates the overall effectiveness of the SPE clean-up step. The results show that the separation strategy is effective for high throughput characterization of proteins from complex proteomic mixtures.

Original language	English
Pages (from-to)	43-51
Number of pages	9
Journal	Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume	787
Issue number	1
DOIs	https://doi.org/10.1016/S1570-0232(02)00616-5
State	Published - 5 Apr 2003
Externally published	Yes

Keywords

Peptides
Proteins
Proteomes

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10.1016/S1570-0232(02)00616-5

Cite this

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title = "Development of a two-dimensional protein-peptide separation protocol for comprehensive proteome measurements",

abstract = "We have developed an effective two-dimensional fractionation protocol of complex proteome mixtures that extends the ability to conduct more comprehensive proteome measurements. A sample containing intact proteins extracted from Saccharomyces cerevisiae was fractionated by liquid phase isoelectric focusing, followed by tryptic digestion and solid-phase extraction (SPE) clean-up and reversed-phase liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS-MS) of the resultant peptides. The clean-up step is designed to desalt the fractions and rid them of urea and ampholytes prior to analysis by LC-MS-MS. Fifty milligrams of protein were separated into 20 fractions by liquid-phase isoelectric focusing, spanning a pH range of 3-10. The effectiveness of the removal of ampholytes was monitored by capillary zone electrophoresis and LC-MS-MS. The ability to analyze all of the 20 fractions without any noticeable decrease in the separation efficiency demonstrates the overall effectiveness of the SPE clean-up step. The results show that the separation strategy is effective for high throughput characterization of proteins from complex proteomic mixtures.",

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author = "Janini, {George M.} and Conrads, {Thomas P.} and Veenstra, {Timothy D.} and Issaq, {Haleem J.}",

note = "Funding Information: The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the US Government. This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-12400.",

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Development of a two-dimensional protein-peptide separation protocol for comprehensive proteome measurements. / Janini, George M.; Conrads, Thomas P.; Veenstra, Timothy D. et al.
In: Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, Vol. 787, No. 1, 05.04.2003, p. 43-51.

Research output: Contribution to journal › Article › peer-review

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AU - Conrads, Thomas P.

AU - Veenstra, Timothy D.

AU - Issaq, Haleem J.

N1 - Funding Information: The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the US Government. This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-12400.

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N2 - We have developed an effective two-dimensional fractionation protocol of complex proteome mixtures that extends the ability to conduct more comprehensive proteome measurements. A sample containing intact proteins extracted from Saccharomyces cerevisiae was fractionated by liquid phase isoelectric focusing, followed by tryptic digestion and solid-phase extraction (SPE) clean-up and reversed-phase liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS-MS) of the resultant peptides. The clean-up step is designed to desalt the fractions and rid them of urea and ampholytes prior to analysis by LC-MS-MS. Fifty milligrams of protein were separated into 20 fractions by liquid-phase isoelectric focusing, spanning a pH range of 3-10. The effectiveness of the removal of ampholytes was monitored by capillary zone electrophoresis and LC-MS-MS. The ability to analyze all of the 20 fractions without any noticeable decrease in the separation efficiency demonstrates the overall effectiveness of the SPE clean-up step. The results show that the separation strategy is effective for high throughput characterization of proteins from complex proteomic mixtures.

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Development of a two-dimensional protein-peptide separation protocol for comprehensive proteome measurements

Abstract

Keywords

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