Biocompare recently featured an article on Bottom-Up Proteomics. I had a chance to follow up with Christie Hunter and expand on some of the questions featured in the article:
1. What sample preparation tools for bottom-up proteomics researchers does SCIEX offer?
A main focus of ours has been on industrializing bottom-up proteomics, from a sample preparation through to obtaining meaningful biological answers. We want to provide full, robust solutions to researchers so they can focus on doing outstanding biology.
From a sample preparation standpoint, we have focused on automation in collaboration with our partners at Beckman Coulter. We have taken well-developed sample prep protocols and automated them on the Biomek NXP Span-8 Workstation (Beckman Coulter). Our first protocol was obviously trypsin digestion as this forms the core of most bottom-up proteomics workflows. SCIEX has a commercially available kit already which includes all the reagents to perform protein denaturation, reduction, alkylation, and trypsin digestion. So, we combined our protein preparation kit with an optimized automation method on the BioMek to provide an out of the box solution that allows researchers to process up to 96 samples in a 5-6 hour time frame with very high reproducibility. Next, we will bolt on additional key workflow modules to serve a broader range of proteomics workflows such as immuno-capture with magnetic beads or isobaric labelling.
2. What types of mass spectrometry instruments, systems, or platforms do you offer for bottom-up proteomics analysis?
We have two main streams of work that we focus on at SCIEX. For discovery proteomics, we are focused on the SWATH® Acquisition technique (a data-independent acquisition technique) for higher throughput comprehensive quantitation. This technique is performed on our TripleTOF® 6600 system which has the dynamic range and MS/MS speed to perform the SWATH workflow. We are now coupling this with microflow chromatography using our NanoLC™ 400 system to enable the analysis of ~150 proteomes a week, a new level of speed and robustness in quantitative proteomics. Watch the community for more information on this workflow soon!
For targeted proteomics, our QTRAP® 6500 system is the key platform because of its very high sensitivity. This Mass Spec has a high degree of flexibility in targeted workflows to provide an effective solution to even the most challenging of quantitative problems. This unique platform is a hybrid triple quadrupole linear ion trap instrument, enabling the instrument to switch from quadrupole mode (MRM - multiple reaction monitoring mode) to ion trap mode in < 1msec. Novel workflows can be constructed using this hybrid platform, such as the MIDAS™ Workflow for fast MRM assay development and MRM identity confirmation.
3. What software tools do you offer or recommend for piecing together mass spectrometry results when using the bottom-up approach?
For processing of our large-scale proteomics data, we have moved our SWATH data processing pipeline to the cloud environment to capitalize on the speed and scale advantages offered by cloud computing. Through our partnership with Illumina on the OneOmics™ Project, we have moved our SWATH processing tools into the BaseSpace Cloud environment. We have apps that will process the raw data, compute differential changes, visualize changes, etc. Data can also be seamlessly pulled into third-party applications, such as iPathwayGuide from Advaita, such that the differential changes can be visualized in the context of pathways or ontologies. Another exciting aspect of the BaseSpace environment is that it serves as an innovation environment, where other researchers can build apps with other functionality and host them for other researchers to use in an easy to use environment.
4. In your opinion, what are the most difficult challenges facing proteomics researchers today when using the bottom-up approach, and what are the most useful tools in this area? Do you expect these to change in the near future?
When you consider the whole samples to knowledge pipeline when it comes to bottom-up proteomics, there are considerations across the board that need to be addressed to truly industrialize proteomics. The field is making great progress on many of these aspects which makes it a truly exciting time to be involved in the field of proteomics. SWATH acquisition continues to grow in impact as the biomarker research field scales up, as evidenced by the innovative new centers that are being established (Stoller Center at the University of Manchester, UK, and the ProCan Center at CMRI in Sydney, Australia). As scale goes up, this will put pressure on all parts of the workflow pipeline, so we will need to continue to push the reproducibility and throughput of sample preparation and analysis and continue to address data processing /interpretation challenges created by large scale.
5. I’ve heard you’re working on a three-part blog series about industrializing proteomics, can you tell us more?
You’ve heard right! Later this week we’re launching the first of a three-part blog series to help educate customers about industrialized proteomics. Each blog will focus on a different topic, acquisition, data processing, and sample preparation. So don’t forget to subscribe to the blog to stay up-to-date on the latest news!