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Mass Spectrometric Analysis of MagneHis™ Purified Proteins

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Abstract

Promega’s MagneHis™ Protein Purification System provides a simple, rapid and reliable method for the purification of polyhistidine-tagged, expressed proteins. Paramagnetic precharged nickel particles (MagneHis™ Ni-Particles) are used to isolate His-tagged proteins directly from a crude cell lysate using either a manual or automated procedure. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry can be used for the identification of His-tagged proteins. We describe an efficient sample preparation method for purified His-tagged proteins resulting in samples that can be directly analyzed by MALDI-TOF mass spectrometry.

Robin Hurst, Gary Kobs and Tonny Johnson

Promega Corporation
Publication Date: 2003

Introduction

In recent years, mass spectrometry (MS) has become an indispensable proteomics tool(1) (2) . Because of their ease of purification and lack of interference with protein function, His-tagged proteins have been combined with mass spectrometry in studies of structure and function(3) , protein-protein interactions(4) , post-translational modifications(5) (6) (7) , molecular weight determination(8) and peptide mapping. Unfortunately, methods normally used for His-tagged protein purification result in the presence of imidazole, and high concentrations of salt and detergents, which interfere with mass spectrometric analysis by causing high backgrounds (Figure 1, Panel A). However, modified washing and elution conditions can decrease the high mass spectrometry backgrounds for His-tagged proteins, as shown in Figure 1, Panels B (– DNA) and C (+ DNA). In this article we describe a method developed for the elution of His-tagged proteins from MagneHis™ Particles that reduces the salt and eliminates imidazole, so that His-tagged proteins can be analyzed more precisely by mass spectrometry.

MALDI-TOF analysis of MagneHis purified His-tagged GFPFigure 1. MALDI-TOF analysis of MagneHis™ purified His-tagged GFP.

A T7 S30-based bacterial cell-free system was used in this experiment. In vitro translated His-tagged GFP was purified using the procedure described in the article. Panel A: His-tagged GFP purified with 0.5M imidazole-containing buffer. Panel B: Control reaction without His-tagged GFP DNA. Panel C: His-tagged GFP DNA purified using the modified wash and elution steps.

MALDI-TOF Analysis of MagneHis™ Purified Proteins

A protocol for preparation of His-tagged proteins for MALDI-TOF analysis, is shown in Table 1. This method can be used for the purification and elution of His-tagged proteins from bacterial cells or cell-free bacterial expression systems. Steps 1–3 describe the purification of His-tagged proteins as directed in the MagneHis™ Protein Purification System Technical Manual, #TM060, while Steps 4–7 describe a modified wash and elution procotol that reduces salt and eliminates imidazole. 

Preparation of MagneHis Purified Proteins for MALDI-TOF Analysis.Table 1. Preparation of MagneHis™ Purified Proteins for MALDI-TOF Analysis.

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Results

A comparison of the standard protocol (TM060 and Table 1, Steps 1–3) and the modified protocol (Table 1, Steps 4–7) for purification/elution of His-tagged Green Fluorescent Protein (His-GFP) expressed in a T7 S30 cell-free expression system and analyzed by MALDI-TOF, is shown in Figure 1, Panels A and C. Panel A is the MALDI-TOF spectrum of the His-GFP purified/eluted with the standard protocol (containing imidazole and high salt concentrations), whereas Panel C shows the MALDI-TOF spectrum of the His-GFP purified/eluted with the modified protocol (Table 1, Steps 4–7). Background peaks are reduced when using the modified protocol in Steps 4–7 of Table 1. SDS-PAGE analysis is shown in Figure 2 (– DNA in Lane 1, + DNA in Lane 2) using the modified protocol for purification/elution. The data in Figure 2 show that using the modified protocol, MALDI-TOF analysis is possible for His-tagged proteins expressed in a bacterial cell-free expression system.

 SDS-PAGE analysis of His-tagged GFP purified from a T7 S30 bacterial cell-free system using the modified protocolFigure 2.  SDS-PAGE analysis of His-tagged GFP purified from a T7 S30 bacterial cell-free system using the modified protocol.

 Lane 1: In vitro coupled transcription and translation of a sample without DNA. Lane 2: In vitro coupled transcription and translation with His-tagged GFP DNA.

Conclusions

The development of a sample preparation method for His-tagged proteins purified using MagneHis™ Protein Purification System can drastically reduce the background levels in MALDI-TOF mass spectrometry analysis. This method can be used for the analysis of His-tagged protein expressed in bacteria in vivo as well as in bacterial cell-free expression systems.

References

  1. Yarmush, M.L. and Jayaraman, A. (2002) Advances in proteomic technologies. Annu. Rev. Biomed. Eng. 4, 349–73.
  2. Hunter, T.C. et al. (2002) The functional proteomics toolbox: Methods and applications. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 782, 165–81.
  3. Sporeno, E. et al. (1994) Production and structural characterization of amino terminally histidine tagged human oncostatin M in E. coli. Cytokine 6, 255–64.
  4. Phan, L. et al. (1998) Identification of a translation initiation factor 3 (eIF3) core complex, conserved in yeast and mammals, that interacts with eIF5. Mol. Cell Biol. 18, 4935–46.
  5. Geoghegan, K.F. et al. (1999) Spontaneous alpha-N-6-phosphogluconoylation of a "His tag" in Escherichia coli: The cause of extra mass of 258 or 178 Da in fusion proteins. Anal. Biochem. 267, 169–84.
  6. Yan, Z. et al. (1999) Mass spectrometric determination of a novel modification of the N-terminus of histidine-tagged proteins expressed in bacteria. Biochem. Biophys. Res. Comm. 259, 271–82.
  7. Yan, Z., Caldwell, G.W. and McDonell, P.A. (1999) Identification of a gluconic acid derivative attached to the N-terminus of histidine-tagged proteins expressed in bacteria. Biochem. Biophys. Res. Comm. 262, 793–800.
  8. Ma, J. et al. (2001) Expression, purification, and MALDI analysis of RPE65. Ophthalmol. Vis. Sci. 42, 1429–35.

How to Cite This Article

Hurst, R. Kobs, G. and Johnson, T. Mass Spectrometric Analysis of MagneHis™ Purified Proteins. [Internet] 2003. [cited: year, month, date]. Available from: http://nld.promega.com/resources/pubhub/enotes/mass-spectrometric-analysis-of-magnehis-purified-proteins/

Hurst, R. Kobs, G. and Johnson, T. Mass Spectrometric Analysis of MagneHis™ Purified Proteins. Promega Corporation Web site. http://nld.promega.com/resources/pubhub/enotes/mass-spectrometric-analysis-of-magnehis-purified-proteins/ Updated 2003. Accessed Month Day, Year.

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Figures

MALDI-TOF analysis of MagneHis purified His-tagged GFPFigure 1. MALDI-TOF analysis of MagneHis™ purified His-tagged GFP.

A T7 S30-based bacterial cell-free system was used in this experiment. In vitro translated His-tagged GFP was purified using the procedure described in the article. Panel A: His-tagged GFP purified with 0.5M imidazole-containing buffer. Panel B: Control reaction without His-tagged GFP DNA. Panel C: His-tagged GFP DNA purified using the modified wash and elution steps.

 SDS-PAGE analysis of His-tagged GFP purified from a T7 S30 bacterial cell-free system using the modified protocolFigure 2.  SDS-PAGE analysis of His-tagged GFP purified from a T7 S30 bacterial cell-free system using the modified protocol.

 Lane 1: In vitro coupled transcription and translation of a sample without DNA. Lane 2: In vitro coupled transcription and translation with His-tagged GFP DNA.

Tables

Preparation of MagneHis Purified Proteins for MALDI-TOF Analysis.Table 1. Preparation of MagneHis™ Purified Proteins for MALDI-TOF Analysis.

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