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Biological Sciences Division
Research Highlights

March 2013

Striking While the Iron Is Hot

Chromatography combined with database search strategy identifies hard-to-find heme proteins

Results spectra and structure
Left: LC-MS results for peptides from bovine cytochrome c. Top: Black line shows the signal for all peptides; red line shows the signal for the single heme c peptide. Bottom: LC-MS chromatogram of the same peptides of bovine cytochrome c after enriching the heme c peptide by HAC. Black line shows the signal for all peptides; red line shows the signal for the single heme c peptide only. The red and black lines coincide, indicating that after HAC, only the heme c peptide remains. Top right: structural model of bovine cytochrome c, showing the heme c group in gray with the heme iron atom in red. Bottom right: schematic of the immobilized histidine ligand attached to a stationary-phase bead (gray sphere), with a model heme c peptide bound to the histidine via the iron atom (small red sphere) of heme group (in red). Enlarge image

Results: Heme c is an important iron-containing post-translational modification found in many proteins. It plays an important role in respiration, metal reduction, and nitrogen fixation, especially anaerobic respiration of environmental microbes. Such bacteria and their c-type cytochromes are studied extensively because of their potential use in bioremediation, microbial fuel cells, and electrosynthesis of valuable biomaterials.

Until recently, these modifications were hard to find using traditional proteomic methods. Scientists at Pacific Northwest National Laboratory combined a heme c tag protein affinity purification strategy called histidine affinity chromatography (HAC) with enhanced database searching. This combination confidently identified heme c peptides in liquid chromatography-tandem mass spectrometry (LC-MS/MS) experiments-by as much as 100-fold in some cases.

Why It Matters: Iron is a critical part of many biological processes; however, it is often not biologically available or it can be toxic in high quantities. So, biological systems have developed intricate methods to use and store iron. Many environmentally important microbes and microbial communities are rich in c-type cytochromes. Combining HAC and data analysis tailored to the unique properties of heme c peptides should enable more detailed study of the role of c-type cytochromes in these microbes and microbial communities.

"Several proteomics studies have analyzed the expression of c-type cytochromes under various conditions," said PNNL postdoctoral researcher Dr. Eric Merkley, and lead author of a paper that appeared in the Journal of Proteome Research. "A shared feature of these studies is that the cytochrome-rich fractions, the cell envelope or extracellular polymeric substance, were purified and explicitly analyzed to efficiently detect cytochromes. Analyses of large-scale proteomics datasets have typically suggested that c-type cytochromes, particularly the heme c peptides, are under-represented."

Methods: The scientists adapted HAC to enrich heme c protein fragments, or peptides, from purified bovine heart cytochrome c, decaheme cytochromes from Sideroxydans lithotrophicus ES-1 and Shewanella oneidensis MR-1, and from mixtures of Escherichia coli peptides with a standard heme peptide at varying ratios. LC-MS/MS analysis of the samples yielded significantly more matches to heme c peptides than in samples not enriched by HAC.

The scientists also developed a proteomics database search strategy that accounts for the unique physicochemical properties of heme c peptides. Combining affinity chromatography and heme-specific informatics yielded 20- to 100-fold increases in the number of peptide-spectrum matches for bovine cytochrome c.

"In addition to demonstrating enrichment of heme c peptides from both simple and complex mixtures, our results indicate that accounting for the double attachment of the heme group is important in identifying heme c peptides, and that the singly charged heme fragment ion is a useful reporter for heme c peptides," said PNNL biochemist Dr. Mary Lipton, who led the study.

What's Next? The scientists are applying and evaluating the methodology for whole-proteome analysis of metal-reducing bacteria. Supplemented by additional fractionation steps, the method could be applied to more complex samples. In addition, HAC combined with mass spectrometric or other methods could become a useful tool for studying the biology of c-type cytochrome biogenesis.

Acknowledgments:

Sponsors: This research was supported by the U.S. Department of Energy Office of Biological and Environmental Research (DOE-BER) Genome Science Program. Significant portions of this work were performed in the Environmental Molecular Sciences Laboratory (EMSL), a national scientific user facility located at PNNL.

User Facility: EMSL

Research Team: Eric Merkley, Mary Lipton, Brian Anderson, Jea Park, Sara Belchik, Liang Shi, Matthew Monroe, and Richard (Dick) Smith, all PNNL.

Research Area: Biological Systems Science

Reference: Merkley ED, BJ Anderson, JH Park, SM Belchik, L Shi, ME Monroe, RD Smith, and MS Lipton. 2012. "Detection and Identification of Heme c-Modified Peptides by Histidine Affinity Chromatography, High-Performance Liquid Chromatography-Mass Spectrometry, and Database Searching." Journal of Proteome Research 11(12):6147-6158. DOI: 10.1021/pr3007914.


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