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Hopkins Group Develops New Method For Identifying Proteins in Microorganisms

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Researchers at the Johns Hopkins Bloomberg School of Public Health have developed a new method for identifying specific proteins in whole cell extracts of microorganisms by using mass spectrometry-based peptide fingerprinting.

The new method involves growing bacteria in a medium that enriches for a protein of interest. The protein of interest can then be analyzed using a MALDI-TOF mass spec without going through the costly and time-consuming process of separating proteins out on a 2D gel, the researchers said.

"Finding a specific target of interest in a mixture of hundreds of proteins can be likened to finding the proverbial needle in the haystack; this task can be performed much faster and more economically if you have more needles — and that's exactly what our method is based on," explained Rolf Halden, an assistant professor in the department of environmental health sciences at Hopkins, and the lead author of a report describing the new method that was published in this month's edition of Applied and Environmental Microbiology.

Halden said Johns Hopkins University is seeking partners who would like to license the new patent-pending methodology.

The Hopkins researchers tested their new methodology using Sphigomonas wittichii strain RW1, a bacterium that is important in cleaning up dioxins, a class of environmental toxins that includes polychlorinated biphenyls, or PCBs.

"Medical waste, or waste in general that is not incinerated at the right temperatures and conditions, can produce a lot of dioxins," said Halden. "RW1 is unique in that it uses dioxin as a food source. It produces an enzyme called dioxin dioxygenase that breaks down the backbone of toxic polychlorinated dibenzo-p-dioxins and dibenzofurans."

Using their new technique, the Hopkins researchers were able to monitor the amount of dioxin dioxygenase present in their batches of bacteria — something that has not previously been done.

"By monitoring the level of protein in the sample, you get a good idea of whether your batch is ready for release, or if you should incubate for another couple of hours," said Halden.

With current techniques, scientists simply measure the amount of biomass of S. wittichii strain RW1 bacteria, but that does not necessarily give a good idea of how much degredative activity the bacteria are capable of, Halden said.

"A lot of genes for degredative activity are located on mobile plasmid units, and sometimes these are lost by the by the bacteria, so if you use biomass, it's not necessarily indicative of the level of [dioxin dioxygenase] enzyme activity," he noted. "There's nothing better than looking for the real enzyme. You get a direct proxy for degredative activity."

Aside from being used as an investigative tool in bioremediation, the new methodology could also be applied to microorganisms that are of clinical interest, Halden said.

"If you are interested in the production of proteins for clinical purposes — for example through the use of genetically engineered E. coli — if the enzyme of interest ionizes well, you could apply the same technique to directly detect how much of the enzyme is present in the bacteria," Halden said.

The key to being able to use the technique to detect specific proteins in whole cell extracts of microorganisms is the ionization capacity of the protein of interest, Halden emphasized. If the protein of interest ionizes well, then it should show up well under mass spec analysis.

"This is an inexpensive way of using these relatively complicated [mass spectrometry] techniques," said Halden. "It demonstrates that peptide mass fingerprinting is not necessarily expensive."

— TSL

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