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.
Growing Up, Moving Out
Exciting times here at Sage Science — we’re moving! We’ve outgrown our current space and will be heading to offices that provide about 50 percent more room for our sizable team. We won’t even have to learn new commutes, as the new space is just downstairs from our old office.
Aside from the extra elbow room, the move is exciting because we’ll have all of our operations in one central space, bringing manufacturing into the same location as the rest of our team for the first time. We’re proud of the fact that we build all of our instruments right here in Beverly, Mass., and it’ll be great for the whole team to see the flurry of activity as new boxes are assembled, tested, and shipped to customers.
The move comes at a time when we are expanding into new markets. Thanks in large part to stellar work from our customers, our Pippin Prep and BluePippin instruments have become the accepted standard for automated DNA size selection, particularly in the NGS space. Now we’re taking our fractionation expertise to the proteomics realm, where automation, precision, and reproducibility can improve results from top-down, bottom-up, and other mass spec workflows. Our new SageELF performs whole-sample fractionation for proteins and DNA, and the BluePippin has been outfitted with fancy new cassettes that allow it to do targeted protein selection.
If you’re ever in the Beverly area, we hope you’ll stop by for a tour of our new digs.
SFAF 2014: Do You Know the Way to Santa Fe?
It seems we just can’t get enough of genomics conferences! This week, Sage Science is proud to be a sponsor of the Sequencing, Finishing, and Analysis in the Future conference hosted by Los Alamos National Laboratory (May 28-30). We’ll be heading to Santa Fe to get our fill of great presentations and posters on what happens after the sequencing run: new methods and best practices for data analysis.
The agenda is built around several useful topics, including: genome sequencing and assembly, finishing tools, genome analysis, and clinical applications of next-gen sequencing. This year’s keynote speakers include Rick Wilson, director of the Genome Institute at Washington University; Deanna Church, senior director of genomics and content at Personalis; and Stephan Schuster, research director at the Singapore Centre on Environmental Life Sciences Engineering.
Talks at SFAF will cover a good mix of sequencing platforms and applications, from metagenomics to clinical to forensic. There will also be many posters — judging from the terrific abstracts, we’ll be spending a lot of time at the poster sessions.
So why is a sample prep company interested in a data analysis meeting? As we’ve carved out our niche in the DNA size selection space, we have found that the people who are most insistent about using Pippin tools are actually the bioinformaticians. They routinely see the difference in quality between assemblies generated from libraries with and without automated size selection and have determined that data from Pippin-sized libraries comes together into cleaner, more accurate assemblies. Never doubt that your sample prep choices have a significant downstream impact!
Microbes in the Spotlight: ASM 2014
Earlier this week, thousands of microbiologists descended on Boston for the annual meeting of the American Society for Microbiology. The Sage Science team was thrilled to be part of the festivities. Many thanks to all of the scientists who visited our booth to learn more about targeted DNA sizing with Pippin and whole-sample fractionation with our new SageELF.
Genomics has steadily increased its presence at ASM over the years. At this meeting, many of the researchers we spoke with were using next-gen sequencers to learn more about their bugs of interest. We heard from many PacBio users interested in improving their average read lengths with Pippin, and from Illumina users eager to construct libraries with multiple insert sizes to generate a better assembly.
There were some general themes at this year’s ASM, including emerging pathogens, big data, and of course the challenge of studying microbes that can’t be cultured. Experts predicted the next pathogen global threats, and one team noted that microbes can live for a week or longer on surfaces found in any airplane. (We’ll be flying in full hazmat suits from now on, thank you very much.) Scientists including George Church and Joachim Messing spoke about the increasing need to merge massive data sets and study them in combination. And several researchers reported on how the “dark matter” of the microbial world, or the organisms that are uncultivable, appear to be impacted by climate change and other stressors. We also heard about the world’s first stool bank designed for fecal transplants, and learned that urine is not bacteria-free, as we always thought it was.
And now it’s back to our regular jobs. It was great to hang out with the microbiology crowd — we’re already looking forward to ASM 2015!
Pippin Enriches for MicroRNAs in Zebrafish Transcriptome Study
Scientists from the Mayo Clinic, Institute for Systems Biology, and University of Illinois recently published a study investigating the effects of vitamin D on microRNA regulation of gene expression in zebrafish. The team used our Pippin Prep together with the NEBNext Multiplex Small RNA Kit from New England Biolabs to enrich their samples for miRNAs.
Lead authors Theodore Craig and Yuji Zhang performed RNA-seq with Illumina to look across the zebrafish transcriptome at which genes’ activity are changed by vitamin D. They found that by the seventh day of development, the zebrafish larvae have 2,500 protein-coding genes that are up- or down-regulated by vitamin D. The authors looked closely at the miRNAs involved in this process, finding that both known and novel miRNAs play a role. “The differentially expressed miRNAs are predicted to potentially alter mRNAs for metabolic enzymes, transcription factors, growth factors, and Jak-STAT signaling,” the authors write.
The scientists hypothesize that the higher number of miRNAs involved by day 7 of development is probably “due to the increased complexity of organs and the need for fine tuning of mRNA expression.”
You can view the paper, “Detection of 1,25-Dihydroxyvitamin D-Regulated miRNAs in Zebrafish by Whole Transcriptome Sequencing,” in the journal Zebrafish.
To learn more about the use of Pippin size selection to enrich miRNA selection with the NEBNext Multiplex Small RNA Kit, check out this app note.