SageELF for Spectral Libraries: An E. coli Example

Timing and proximity can work wonders when it comes to scientific collaborations.

As we were first developing the SageELF, a unique protein sample fractionation device, we discovered by chance that it fit perfectly into an initiative spearheaded by Jeff Silva of Cell Signaling Technologies — our neighbors here in Beverly, Mass.

At the time, Jeff and collaborating researchers from Gordon College, Waters Corp., and the Ionomix Initiative were interested in creating spectral peptide libraries for E. coli. The idea was to test a concept in which a comprehensive mass spec analysis of the E. coli proteome could be performed to provide researchers with a useful resource for future studies. With such a spectral library, the idea went, any protein scientist working with E. coli could do a preliminary scan, run a quick comparison against the spectra on the list and check those off, and then devote mass spec analytical resources to identifying proteins not in the library.

To get a comprehensive snapshot of the organism’s proteome, scientists at Gordon College, led by Dr. Justin Topp, created a “mega-sample” by growing E. coli under more than 60 different conditions, including growth phases, media types, and stress conditions. Then they extracted and pooled the proteins.

Enter the SageELF. The protein sample was separated by size into 12 fractions, upstream of trypsin digestion and LC/MS. This vastly reduced the complexity of the sample and allowed scientists at Waters to test the reaches of their analytical sensitivity.

After ELF fractionation, the sample complexity was even further reduced by a second dimension of separation (reversed-phase LC with the Waters nanoAcquity UPLC) before moving the samples to the Waters SYNAPT-G2S HDMS system to provide deep analytical coverage.

Of course, the name of game with complex mixtures is to find strategies for deconvolving isobaric peaks. Michael Nold, proteomics group leader at Waters, and Manor Askenazi of the Ionomix Initiative provided the software and analysis expertise to tackle the challenge.

How did we do? The team estimated that we identified up to 80 percent of the E. coli proteome — not bad for a first shot. For our part, we feel the SageELF could do much better now that development has been completed; we are getting much better separation in the fractions (i.e. less overlap in adjacent samples). If you’d like to check out the scientific poster we presented at 2013’s ASMS, you can view it here.

We’d like to thank everyone on the team, especially Jeff, for working with us and giving us the opportunity to demonstrate the SageELF. Since we got so much out of this serendipitous neighborhood encounter, we’d like to make sure everyone knows about another great local networking opportunity: the Life Sciences Consortium of the North Shore (of Massachusetts). Our founder and board member Gary Magnant was instrumental in creating this consortium, and we hope that it helps other companies have the same kind of luck we had with Jeff and his team.

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