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Northwestern's Kelleher and Colleagues Introduce Protease for 'Middle-Down' Proteomics

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A team led by Northwestern University researcher Neil Kelleher has developed a new protein digestion technique for generating large peptide fragments.

The method, which was detailed in a paper published this week in Nature Methods, uses outer membrane protease T – OmpT – to produce peptides larger than those generated by conventional trypsin digestion, enabling researchers to pursue a "middle-down" approach that could combine benefits of both top-down and bottom-up proteomics.

Bottom-up proteomics – which uses trypsin to digest proteins into small peptides more amenable to mass spec analysis – has traditionally dominated the field. This approach, however, has drawbacks, including difficulty assigning peptides and the loss of information on post-translational modifications and isoforms.

Partially in response to these issues, interest in top-down proteomics – which looks at intact proteins – has grown in recent years. However, this technique remains technically challenging and declines in effectiveness when dealing with high-mass proteins.

A middle-down proteomics approach could prove an effective compromise between the two methods, employing digestion to create peptides that can be analyzed relatively easily by mass spec but which are large enough to retain richer sequence and PTM information.

Middle-down isn't a new concept, noted Kelleher, who is a leading top-down researcher (PM 11/4/2011). However, he told ProteoMonitor, it's been a difficult idea to implement due to the lack of digestion reagents for generating large peptides from a complex proteome. While such digestions are frequently done for targeted work – as in x-ray crystallography, for instance – "no one has shown it in a complex mixture in a proteomic context," he said.

"It's hard – people will say, 'Why not try a chemical digestion [method]?' But [with] chemical methods it's a nightmare of side chain reactions and oxidations," Kelleher said. "Or, if you take trypsin and try [to partially digest the sample]… you end up with this sort of sea of complexity from the limited digestion."

Ultimately, the scientists turned to the brain where, informed by the work of their study co-author Jonathan Sweedler – a University of Illinois researcher specializing in neuropeptidomics – they looked at proteases involved in generating neuropeptides.

"Neuropeptides are produced by proteases that recognize two amino acids instead of one," Kelleher said. Because such proteases will cut only at sites where both amino acids are present they will typically generate larger peptides than a protease like trypsin that recognizes a single peptide.

In addition to its di-basic cleavage properties, OmpT exhibited a variety of other qualities desirable in a protease for use in proteomics research, he said, including good thermodynamic stability, resistance to soaps and surfactants, and good kinetics.

The researchers have a patent on the enzyme and are potentially interested in licensing it, Kelleher said, suggesting that it would probably make for a viable, if somewhat niche, reagent product.

"It would be a small little thing, I think, and we'll have to see its performance over the long run and whether there would be sustained interest in it as a product, but it could certainly support a product," he said.

Although middle-down proteomics isn't widely practiced at present, Kelleher said that the current mass spec landscape suggests the technique is poised to take off. While top-down proteomics has typically been the purview of high-powered FT-ICR machines and, more recently, Thermo Fisher Scientific's Orbitrap Elite instrument, middle-down is amenable to a wide variety of systems, he said.

"It's actually a resonance point," Kelleher said. "Orbitraps, and FT-ICRs, and time-of-flight [instruments] can all perform better at this higher mass. Three thousand to 15,000 Daltons is a nice sweet spot for the [performance of the] latest instruments. It brings a lot more kinds of instruments into play."

The FT-ICR market has traditionally been dominated by Bruker with Thermo Fisher also providing some offerings in this area, but most major vendors, including Agilent, Waters, and AB Sciex, offer time-of-flight machines.

Kelleher noted that middle-down is also poised to draw interest due to the growing use of electron transfer dissociation as a mass spec fragmentation technique.

"Electron-based fragmentation methods like ETD work better if you have more highly charged species and larger species, but there's been no protease in proteomics to create [peptides] that have high – six-plus, seven-plus – charge states," he said. "Now you have the possibility of [creating such peptides], and that really functions well [with ETD]. You can sequence almost the whole peptide because the more charges you have on the ion the more electron affinity you have."

Aside from shotgun experiments, the protease could also prove popular for targeted analyses, Kelleher said, particularly where researchers or clinicians want to be certain they are measuring a particular form of a protein.

"Say you're interested in a protein and [particular modification] sites and you can't map it using other proteases," he said. "This would be a great option because you can [analyze] bigger chunks."

In the Nature Methods paper, the researchers used the OmpT-based method to characterize the 20 kDa to 100 kDa protein fraction of the HeLa cell proteome, identifying 3,697 unique peptides and 1,038 unique proteins via analysis on either a Thermo Scientific LTQ-FT instrument or a Thermo Scientific Orbitrap Elite.

They used an upfront gel-free separation step to isolate the 20 kDa to 100 kDa fraction so that they could concentrate on the high mass proteins with which top-down proteomics still struggles, Kelleher said. The average size of the peptides generated by digestion with OmpT was 6.3 kDa.

"We only looked at peptides in the zero to 15 kDa range because for now that's the resonance [point] that a lot of people on a lot of diverse instruments can focus on," he added.

Kelleher noted that his lab is able to perform top-down analysis on proteins larger than 70 kDA, but that such efforts are very complicated technically.

"This [middle-down approach] is a great mechanism to get [researchers] ready to start analyzing larger piece of proteins," he said. "It’s a stepping stone for the field as it drifts [toward more top-down work.]"

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