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PNNL Top-Down Study IDs 1600-plus Salmonella Proteoforms Including Previously Undetected PTM

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A team led by researchers at Pacific Northwest National Laboratory has devised a streamlined top-down proteomics approach that they have used to profile the pathogen Salmonella Typhimurium.

Using the workflow, which features a single dimension of chromatographic separation as opposed to the multi-dimensional separation procedures typical in top-down work, the researchers identified in Salmonella a post-translational modification – S-cysteinylation – not previously detected in gram-negative bacteria.

S-cysteinylation is a form of protein S-thiolation, a modification involved in processes including cell response to oxidative stress. Presenting their results in a paper published this week in the Proceedings of the National Academy of Sciences, the PNNL team and their co-authors observed that under infection-like conditions, Salmonella switches from S-glutathionylation to the newly identified S-cysteinylation.

This shift, they hypothesized, is designed to help the organism conserve energy amidst the stresses of the infection process, moving from using the large glutathione molecule for S-thiolation to the smaller, and thus less energy-intensive, cysteine molecule.

The findings are an example of the power of top-down proteomics, Ljiljana Pasa-Tolic, a PNNL researcher and author on the PNAS paper, told ProteoMonitor. Top down methods allow "you to work in this discovery mode of operation, where you can find things that you don't know about," she said.

Bottom-up, peptide-based proteomics has dominated the field for the last decade, but interest in top-down methods has grown as improvements in instrumentation have made analysis of intact proteins easier and researchers have become increasingly aware of the importance of protein isoforms and post-translational modifications.

As Pasa-Tolic noted, however, at this point top-down work remains considerably more challenging than bottom-up efforts. While bottom-up researchers can now consistently identify in the range of 10,000 proteins in a given experiment, identifying in the range of 1,000 proteins and 3,000 proteoforms, as Northwestern University researcher Neil Kelleher demonstrated in a 2011 Nature paper (PM 11/4/2011), is considered a significant achievement for a top-down experiment.

"Every step is more difficult," Pasa-Tolic said. Separating them is more difficult; doing tandem mass spec becomes very difficult as the mass increases; and then the whole bioinformatics becomes more difficult – assignment of identity and assignment of the modification sites – as the proteins get larger."

Large-scale, top-down work has typically relied on extensive separation upfront of mass spec analysis. Kelleher's Nature study, for instance, used three steps: solution isoelectric focusing, gel-eluted liquid fraction entrapment electrophoresis, and nanocapillary liquid chromatography.

Pasa-Tolic and her colleagues, on the other hand, sought to reduce the amount of upfront separation with the aim of reducing run times and enabling the use of smaller sample sizes. Using single-dimension UPLC coupled to a Thermo Fisher Scientific LTQ Orbitrap Velos, they identified in Salmonella 563 proteins and 1,665 proteoforms, representing what they said was the largest prokaryotic top-down dataset to date.

"Our lab over the years has developed quite an expertise in the area of separations, and so we were able to actually achieve high enough resolution in a single-stage separation, and this allowed us to reduce the amount of material we needed for analysis quite significantly," Pasa-Tolic said, noting that with the system the researchers could analyze sample amounts as low one microgram.

"That was one of our leading goals [in the study]," she added. "To scale this down so we could reduce the sample requirements. Because [high sample volume requirements] just by definition will exclude a number of applications."

The researchers also varied the bioinformatics portion of their workflow, using for their analysis the MS-Align+ algorithm developed by their co-author, University of California, San Diego researcher Pavel Pevzner, instead of the more commonly used ProSight PTM software, which was developed by Kelleher and is sold by Thermo Fisher.

The MS-Align+ program uses a spectral alignment algorithm for making identifications, which, said Pasa-Tolic, offers advantages in terms of looking for previously unidentified modifications such as the Salmonella S-cysteinylation that she and her colleagues observed.

With improvements in top-down proteomics workflows, the technique is moving toward becoming "a more everyday technique," she said, noting that especially in the case of microbial organisms researchers are able to achieve significant proteome coverage. She cited work presented by her group at this year's American Society for Mass Spectrometry Conference on Top Down Mass Spectrometry in January in which they identified in Escherichia coli 1,249 gene products and more than 4,000 proteoforms. That study, which the researchers are currently writing up for publication, represents, to their knowledge, the largest top-down proteomics dataset generated to date, Pasa-Tolic said.

Despite these advances, however, significant challenges remain in terms of broadening the technique's usefulness, she said. For instance, current top-down workflows are limited to smaller proteins – in the 40 kD and below range, typically. This, she said, was due primarily to limitations in mass spec instrumentation and in fragmentation techniques. Separation methods are also in need of additional improvement, she said.

Work is underway on tackling these problems, she noted, citing in particular work by University of Texas researcher Jennifer Brodbelt on using ultraviolet photodissociation for fragmenting proteins for top-down analysis.

Brodbelt and her colleagues "have shown that they can achieve very high coverage looking at intact proteins [using UV photodissociation]," Pasa-Tolic. "So I think that it would certainly be an option in the future to implement something like that in a commercial [mass spec] instrument."

She also noted work ongoing at PNNL on a 21 Tesla Fourier transform ion cyclotron resonance mass spectrometer that she said would be the highest field FT-ICR instrument available when it comes online next year.

"FT-ICR ultimately has the highest mass resolving power of mass spectrometers, which is important for being able to distinguish isotopes within the envelope of the highly charged large proteins," she said. "So to extend top down beyond 100 [kD] or so, you probably at present have the best chance using this high field FT-ICR instrument."

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