Combining chromatography, proteomics and database searching identifies hard-to-find heme proteins

May 14, 2013 by Kristin Manke
Combining chromatography, proteomics and database searching identifies hard-to-find heme proteins
Detection of heme c peptides (red) and all peptides (black) by LC-MS before (top) and after (bottom) HAC.

(Phys.org) —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 for its use and storage. Scientists at DOE's Pacific Northwest National Laboratory combined chromatography, proteomics and a database search strategy to find higher numbers of modified iron-containing protein fragments (called peptides) that play an important role in respiration, metal reduction and nitrogen fixation by environmental microbes. The microbes containing these proteins are being studied because of their potential use in microbial fuel cells and electrosynthesis of valuable biomaterials.

The modifying group of atoms, called heme c, is an important iron-containing post-translational modification found in many proteins. Until recently, it was hard to find. The PNNL scientists combined a heme c protein strategy called histidine affinity chromatography (HAC) with enhanced database searching. This combination confidently identified heme c peptides in -tandem mass spectrometry (LC-MS/MS) experiments by as much as 100-fold in some cases. They used proteomics capabilities housed at DOE's EMSL, located at PNNL.

"Several proteomics studies have analyzed the expression of c-type cytochromes under various conditions," said PNNL researcher Dr. Eric Merkley, 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."

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 .

The scientists adapted HAC to enrich heme c , 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.

They 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.

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.

Explore further: Researchers successfully clone adult human stem cells

add to favorites email to friend print save as pdf

Related Stories

Unique E. coli protein may be not after all

Jan 03, 2012

A bacterial protein recently thought to be a unique mechanism for utilizing iron may not be after all. Researchers from the University of Georgia, the Fellowship for Interpretation of Genomes, the University of Oklahoma and ...

Clustering is key to lighting up the dark proteome

Aug 04, 2011

(PhysOrg.com) -- A new approach that organizes previously unused mass spectra from proteomics studies gives scientists the ability to use these spectra to gain more information about proteins in a wide range ...

Heme channel found

Dec 17, 2009

In some ways a cell in your body or an organelle in that cell is like an ancient walled town. Life inside either depends critically on the intelligence of the gatekeepers.

Recommended for you

Researchers successfully clone adult human stem cells

Apr 18, 2014

(Phys.org) —An international team of researchers, led by Robert Lanza, of Advanced Cell Technology, has announced that they have performed the first successful cloning of adult human skin cells into stem ...

Researchers develop new model of cellular movement

Apr 18, 2014

(Phys.org) —Cell movement plays an important role in a host of biological functions from embryonic development to repairing wounded tissue. It also enables cancer cells to break free from their sites of ...

For resetting circadian rhythms, neural cooperation is key

Apr 17, 2014

Fruit flies are pretty predictable when it comes to scheduling their days, with peaks of activity at dawn and dusk and rest times in between. Now, researchers reporting in the Cell Press journal Cell Reports on April 17th h ...

User comments : 0

More news stories

Biologists help solve fungi mysteries

(Phys.org) —A new genetic analysis revealing the previously unknown biodiversity and distribution of thousands of fungi in North America might also reveal a previously underappreciated contributor to climate ...

Researchers successfully clone adult human stem cells

(Phys.org) —An international team of researchers, led by Robert Lanza, of Advanced Cell Technology, has announced that they have performed the first successful cloning of adult human skin cells into stem ...

NASA's space station Robonaut finally getting legs

Robonaut, the first out-of-this-world humanoid, is finally getting its space legs. For three years, Robonaut has had to manage from the waist up. This new pair of legs means the experimental robot—now stuck ...

Ex-Apple chief plans mobile phone for India

Former Apple chief executive John Sculley, whose marketing skills helped bring the personal computer to desktops worldwide, says he plans to launch a mobile phone in India to exploit its still largely untapped ...

Filipino tests negative for Middle East virus

A Filipino nurse who tested positive for the Middle East virus has been found free of infection in a subsequent examination after he returned home, Philippine health officials said Saturday.

Egypt archaeologists find ancient writer's tomb

Egypt's minister of antiquities says a team of Spanish archaeologists has discovered two tombs in the southern part of the country, one of them belonging to a writer and containing a trove of artifacts including reed pens ...

Airbnb rental site raises $450 mn

Online lodging listings website Airbnb inked a $450 million funding deal with investors led by TPG, a source close to the matter said Friday.