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Hundreds of attendees packed the Hyatt Regency on June 15 for the launch of the SCIEX TripleTOF 6600 System with SWATH® Acquisition 2.0.
The "Next-Generation Proteomics Platform" enables the quantitation of thousands of proteins across large sample sets with MRM accuracy and reproducibility. Researchers can now dig deeper into the proteome to find meaningful protein relationships faster than ever before.
Significant advances in SWATH® 2.0 are made possible by the power of the new TripleTOF 6600. It delivers 5 orders of linear dynamic range with improved coverage, mass range up to 2250 m/z, faster acquisition rates and outstanding mass accuracy and stability.
"Variable windows in SWATH® 2.0 let you customize and fine tune your data acquisition approach," noted Dr. Christie Hunter, Director of Omics Applications at SCIEX. Previously, SWATH® provided 25 Da Q1 windows. SWATH® 2.0 now supports up to 200 variable windows, enabling significantly smaller Q1 windows to match the samples being analyzed.
"SWATH® 2.0 lets you easily optimize your windows strategy," Dr. Hunter said. "You can use small windows when there's a lot of peptide density, and to cover the same mass range, you can open up the window size at higher molecular masses to access the precursors there."
Window size can be defined through a text file that is loaded into the method builder, which then automatically builds the SWATH® acquisition method.
For more information on the power of SWATH® 2.0, view Dr. Hunter's presentation.
Check out these insightful presentations from ASMS 2014:
In his workshop, Building High-Quality Libraries for SWATH® MS, Dr. Yansheng Liu (Postdoctoral Researcher, Institute of Molecular Systems Biology, ETH, Zurich, Switzerland) described a 5-step approach that begins with data preparation and acquisition using fractionation, followed by data format conversion, routine DB search, spectral library generation with RT calibration, and data table generation that is compatible with specific software tools.
The data presented includes spectral m/z, RT values and fragment intensities. Dr. Liu also cited work on a pan human MS-AC library of 10,000 proteins to which contributors can modify and add.
Jakob Vowinckel (Research Associate, University of Cambridge, Department of Biochemistry, Cambridge, UK), in his work with yeast and how its metabolism reacts to stress, outlined his approach to achieve higher throughput acquisition with SWATH®. He described in his presentation Microflow LC SWATH® Acquisition for Higher Throughput how high mass spectrometer performance combined with robust microLC (versus nanoLC ) allowed the processing of the requisite large number of samples while maintaining excellent quantitation and data quality.
Combining MS1 and SWATH® Data was presented by Brad Gibson (Director of Chemistry and Mass Spectrometry Core, Buck Institute for Research on Aging, Novato, CA), who investigated whether MS1 data filtering could be integrated into SWATH® Acquisition for enhanced data quality. Collaborating with a group at Purdue University, he developed methods that would use fragmented ions as well as M+1 and M+2 ions in the MS1 acquisition as part of the SWATH® run.
He found that in small samples, the MS1 signal was often better than ion data, "but SWATH® is clearly better in more complex samples," he said.
Gibson, who has tested SWATH® acquisition with variable windows, found that variable windows could be very effective in eliminating MS2 interference. "That has real value", he noted, telling the audience "I encourage you to try using variable SWATH® windows on a 6600 – it's working awesomely."
Andreas Otto (Ph.D., University of Greifswald, Institute for Microbiology, Greifswald, Germany) focuses on quantitative microbial proteomics and is researching the increasing resistance of bacteria to antibodies. In his workshop, SWATH® vs. 15N Labeling - Less Time, More Information, he presented his investigation of SWATH® acquisition as an alternative label-free data acquisition method.
"Bacteria is label-intensive and cost-intensive, and not all bacteria are amenable to labeling," Dr. Otto explained. His team ran a target sample using 15N labeling, then compared results with SWATH® data acquired on a TripleTOF 5600.
A total of 1132 proteins were found using a shotgun approach, versus 832 using SWATH® acquisition. Approximately 80% of the proteins in the spectral library were quantified in a 100-minute run, versus taking days of measurements using 15N labeling for soluble proteins.
"SWATH® acquisition provides a great opportunity to do differential analysis, " he told the audience. "It's easily implemented in large studies and you get data without missing values for approximately 1,000 proteins per 100-minute run," Dr. Otto said, adding: "This is a really cool number for biological studies."
In the workshop, SWATH® vs. MRM - How to Decide?, Brett Larsen (Mass Spectrometer Coordinator, Lunenfeld-Tanenbaum Research Institute, Toronto, Canada) explained how his team has implemented both SWATH® and MRM strategies in its research, and noted that there are clear advantages with each.
While MRM is more sensitive, it offers disparate coverage of the mass range and takes a "fair amount of development time because we have low-level signals," he noted. He said that SWATH® acquisition, on the other hand, "is very easy and robust, with easy method development."
Larsen highlighted the value of SWATH® variable windows, including the capability to have variable window sizes from 1 Da to 150 Da or larger across the mass range and still get very accurate extraction afterwards. He found that different window sizes don't have an effect on quantitation within a 10% variation across runs, "and that's great," he commented.
"Establishing a method over a course of sample, despite having variable windows, won't affect sensitivity," he said.
Larsen also described how "blending" SWATH® acquisition and MRM can be a very effective strategy.
"You can keep a set of the 20 most interesting peptides in your assay set, then use SWATH® variable windows to cover all the spaces in between to achieve good quantitation across the whole sample set - but still get high specificity on a given number of targets," he explained.
Dr. Larsen suggested that for some samples, MRM may not even be required. Subsets of peptides that are "fairly easy to detect anyway" can be handled in regular SWATH® windows, while peptides that are "trickier" to detect can be focused in 1 Da-size windows.
"It's not clear if we'll be doing much MRM in the future, or just use the SWATH® variable windows approach - especially if we can get up to 200 windows for a particular assay with a moderate cycle time. We'll probably focus more on variable windows SWATH® going forward," he added.
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