A Robust Two-Dimensional Separation for Top-Down Tandem Mass Spectrometry of the Low-Mass Proteome

Ji Eun Lee; John F. Kellie; John C. Tran; Jeremiah D. Tipton; Adam D. Catherman; Haylee M. Thomas; Dorothy R. Ahlf; Kenneth R. Durbin; Adaikkalam Vellaichamy; Ioanna Ntai; Alan G. Marshall; Neil L. Kelleher

doi:10.1016/j.jasms.2009.08.001

A Robust Two-Dimensional Separation for Top-Down Tandem Mass Spectrometry of the Low-Mass Proteome

Ji Eun Lee, John F. Kellie, John C. Tran, Jeremiah D. Tipton, Adam D. Catherman, Haylee M. Thomas, Dorothy R. Ahlf, Kenneth R. Durbin, Adaikkalam Vellaichamy, Ioanna Ntai, Alan G. Marshall, Neil L. Kelleher^*

^*Corresponding author for this work

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

86 Scopus citations

Abstract

For fractionation of intact proteins by molecular weight (MW), a sharply improved two-dimensional (2D) separation is presented to drive reproducible and robust fractionation before top-down mass spectrometry of complex mixtures. The "GELFrEE" (i.e., gel-eluted liquid fraction entrapment electrophoresis) approach is implemented by use of Tris-glycine and Tris-tricine gel systems applied to human cytosolic and nuclear extracts from HeLa S3 cells, to achieve a MW-based fractionation of proteins from 5 to >100 kDa in 1 h. For top-down tandem mass spectroscopy (MS/MS) of the low-mass proteome (5-25 kDa), between 5 and 8 gel-elution (GE) fractions are sampled by nanocapillary-LC-MS/MS with 12 or 14.5 tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Single injections give about 40 detectable proteins, about half of which yield automated ProSight identifications. Reproducibility metrics of the system are presented, along with comparative analysis of protein targets in mitotic versus asynchronous cells. We forward this basic 2D approach to facilitate wider implementation of top-down mass spectrometry and a variety of other protein separation and/or characterization approaches.

Original language	English (US)
Pages (from-to)	2183-2191
Number of pages	9
Journal	Journal of the American Society for Mass Spectrometry
Volume	20
Issue number	12
DOIs	https://doi.org/10.1016/j.jasms.2009.08.001
State	Published - Dec 2009

Funding

The authors thank Christopher L. Hendrickson, Mark R. Emmett, John P. Quinn, and Greg T. Blakney of the National High Magnetic Field Laboratory for helpful advice and discussion, and the National Science Foundation (DMR-06-54118) for supporting this national facility. The authors also thank Mingxi Li for helping with sample preparations. The laboratory of NLK was supported by the Packard Foundation, the Sloan Foundation, and the National Institutes of Health (GM-067193-07) and JL was supported by the UIUC Neuroproteomics Center on Cell-Cell Signaling supported by the National Institute on Drug Abuse (P30 DA-018310-06).

ASJC Scopus subject areas

Structural Biology
Spectroscopy

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10.1016/j.jasms.2009.08.001

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Lee, J. E., Kellie, J. F., Tran, J. C., Tipton, J. D., Catherman, A. D., Thomas, H. M., Ahlf, D. R., Durbin, K. R., Vellaichamy, A., Ntai, I., Marshall, A. G., & Kelleher, N. L. (2009). A Robust Two-Dimensional Separation for Top-Down Tandem Mass Spectrometry of the Low-Mass Proteome. Journal of the American Society for Mass Spectrometry, 20(12), 2183-2191. https://doi.org/10.1016/j.jasms.2009.08.001

@article{b90721acc7e94d7cb00d75e0fff09c09,

title = "A Robust Two-Dimensional Separation for Top-Down Tandem Mass Spectrometry of the Low-Mass Proteome",

abstract = "For fractionation of intact proteins by molecular weight (MW), a sharply improved two-dimensional (2D) separation is presented to drive reproducible and robust fractionation before top-down mass spectrometry of complex mixtures. The {"}GELFrEE{"} (i.e., gel-eluted liquid fraction entrapment electrophoresis) approach is implemented by use of Tris-glycine and Tris-tricine gel systems applied to human cytosolic and nuclear extracts from HeLa S3 cells, to achieve a MW-based fractionation of proteins from 5 to >100 kDa in 1 h. For top-down tandem mass spectroscopy (MS/MS) of the low-mass proteome (5-25 kDa), between 5 and 8 gel-elution (GE) fractions are sampled by nanocapillary-LC-MS/MS with 12 or 14.5 tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Single injections give about 40 detectable proteins, about half of which yield automated ProSight identifications. Reproducibility metrics of the system are presented, along with comparative analysis of protein targets in mitotic versus asynchronous cells. We forward this basic 2D approach to facilitate wider implementation of top-down mass spectrometry and a variety of other protein separation and/or characterization approaches.",

author = "Lee, {Ji Eun} and Kellie, {John F.} and Tran, {John C.} and Tipton, {Jeremiah D.} and Catherman, {Adam D.} and Thomas, {Haylee M.} and Ahlf, {Dorothy R.} and Durbin, {Kenneth R.} and Adaikkalam Vellaichamy and Ioanna Ntai and Marshall, {Alan G.} and Kelleher, {Neil L.}",

note = "Funding Information: The authors thank Christopher L. Hendrickson, Mark R. Emmett, John P. Quinn, and Greg T. Blakney of the National High Magnetic Field Laboratory for helpful advice and discussion, and the National Science Foundation (DMR-06-54118) for supporting this national facility. The authors also thank Mingxi Li for helping with sample preparations. The laboratory of NLK was supported by the Packard Foundation, the Sloan Foundation, and the National Institutes of Health (GM-067193-07) and JL was supported by the UIUC Neuroproteomics Center on Cell-Cell Signaling supported by the National Institute on Drug Abuse (P30 DA-018310-06). ",

year = "2009",

month = dec,

doi = "10.1016/j.jasms.2009.08.001",

language = "English (US)",

volume = "20",

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journal = "Journal of the American Society for Mass Spectrometry",

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Lee, JE, Kellie, JF, Tran, JC, Tipton, JD, Catherman, AD, Thomas, HM, Ahlf, DR, Durbin, KR, Vellaichamy, A, Ntai, I, Marshall, AG & Kelleher, NL 2009, 'A Robust Two-Dimensional Separation for Top-Down Tandem Mass Spectrometry of the Low-Mass Proteome', Journal of the American Society for Mass Spectrometry, vol. 20, no. 12, pp. 2183-2191. https://doi.org/10.1016/j.jasms.2009.08.001

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T1 - A Robust Two-Dimensional Separation for Top-Down Tandem Mass Spectrometry of the Low-Mass Proteome

AU - Lee, Ji Eun

AU - Kellie, John F.

AU - Tran, John C.

AU - Tipton, Jeremiah D.

AU - Catherman, Adam D.

AU - Thomas, Haylee M.

AU - Ahlf, Dorothy R.

AU - Durbin, Kenneth R.

AU - Vellaichamy, Adaikkalam

AU - Ntai, Ioanna

AU - Marshall, Alan G.

AU - Kelleher, Neil L.

N1 - Funding Information: The authors thank Christopher L. Hendrickson, Mark R. Emmett, John P. Quinn, and Greg T. Blakney of the National High Magnetic Field Laboratory for helpful advice and discussion, and the National Science Foundation (DMR-06-54118) for supporting this national facility. The authors also thank Mingxi Li for helping with sample preparations. The laboratory of NLK was supported by the Packard Foundation, the Sloan Foundation, and the National Institutes of Health (GM-067193-07) and JL was supported by the UIUC Neuroproteomics Center on Cell-Cell Signaling supported by the National Institute on Drug Abuse (P30 DA-018310-06).

PY - 2009/12

Y1 - 2009/12

N2 - For fractionation of intact proteins by molecular weight (MW), a sharply improved two-dimensional (2D) separation is presented to drive reproducible and robust fractionation before top-down mass spectrometry of complex mixtures. The "GELFrEE" (i.e., gel-eluted liquid fraction entrapment electrophoresis) approach is implemented by use of Tris-glycine and Tris-tricine gel systems applied to human cytosolic and nuclear extracts from HeLa S3 cells, to achieve a MW-based fractionation of proteins from 5 to >100 kDa in 1 h. For top-down tandem mass spectroscopy (MS/MS) of the low-mass proteome (5-25 kDa), between 5 and 8 gel-elution (GE) fractions are sampled by nanocapillary-LC-MS/MS with 12 or 14.5 tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Single injections give about 40 detectable proteins, about half of which yield automated ProSight identifications. Reproducibility metrics of the system are presented, along with comparative analysis of protein targets in mitotic versus asynchronous cells. We forward this basic 2D approach to facilitate wider implementation of top-down mass spectrometry and a variety of other protein separation and/or characterization approaches.

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A Robust Two-Dimensional Separation for Top-Down Tandem Mass Spectrometry of the Low-Mass Proteome

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