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A Mine of Quantitative Proteomic Information

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Prof Dr. Ruedi Aebersold, Head of the Department of Biology, ETH Zurich

The Aebersold group at ETH Zurich focuses on proteomics research, including the development of techniques to study the proteome as an integrated entity. In collaboration with SCIEX, the group established SWATH® Acquisition mass spectrometry, a data-independent acquisition (DIA) method capable of fragmenting multiple peptide species concurrently. The resulting comprehensive data set can be retrospectively re-mined, enabling maximum benefit to be derived from any study.

The rapidly growing field of proteomics is the focus of the Aebersold group’s research. As part of its research program, the group is developing new techniques for the analysis of the proteome, with MS its preferred analytical method. Professor Ruedi Aebersold, Head of the Department of Biology, explained, “There are two ways to study proteins; affinity reagent- based proteomic approaches and MS. Antibody-based reagents work well but, other than for the human proteome, the antibody source is not rich. There are virtually no systematic antibody panels available for species such as yeast, mice or flies. For these species, and human proteomics, MS is the method of choice. MS is the most generic and unbiased tool presently available for the analysis of proteins and proteomes. It allows de novo identification of proteins and, to a degree, the extent of modification. We can even get an idea of how proteins fold and interact.”

He continued, “We have been using MS for a long time, and the technology has advanced considerably over the years. The instruments are far more sensitive now, with higher resolution and faster scanning capabilities. As a result, the methods used for protein analysis have changed a lot.

One important milestone was the development of quantitative techniques allowing the determination of protein abundance, and another was the improvement in software, where sophisticated statistical tools indicate whether a result is true or false, for example, if a protein has been correctly identified or not.

The third big advance was the transition from data-dependent analysis, or shotgun sequencing, to data-independent SWATH Acquisition. This transition is still ongoing, but I believe that the application of SWATH-MS will transform proteomics, particularly for large-scale studies.

The disadvantage of data-dependent analysis is that repeat analyses can be inconsistent, purely because the instrument sampled a different subset of peptides from a complex sample matrix. SWATH alleviates this to a great extent; instead of fragmenting a single peptide species at a time, multiple peptide species are fragmented concurrently. This is a great step forward because you don’t miss any peptides and the random element is removed. The challenge then is data analysis, because the method generates convoluted spectra consisting of fragment ions from different precursors, which cannot be meaningfully analyzed by conventional search engines. DIA is not a new concept, but initially, it did not take off because data acquisition was either too slow or not up to the task, and the problem of evaluating convoluted fragment ion spectra had generally not been solved.

However, when I saw the SCIEX TripleTOF® 5600+, it was immediately clear that this instrument could perform DIA in a way that eliminates the data acquisition issues. Also, the data analysis challenges could be eradicated by the implementation of a targeted strategy. Together with SCIEX, we went on to implement and patent the SWATH technique, which has now been widely adopted.”

“SWATH is ideal for proteomics, allowing us to investigate, for example, how genomic variability is translated into phenotypes, and go on to perform biomarker studies. For the first time, we can measure hundreds of similar samples reliably and with moderate effort, cells of exactly the same type but at different states, samples from a large number of individuals, or from different tissues from one individual, and make meaningful comparisons. With these large cohorts, minimizing variability due to sample preparation is vital, and so we use Pressure Cycling Technology (PCT). PCT is a very fast sample preparation method that eliminates much of the inherent variability, generating reproducible peptide samples. It also allows us to run up to 96 samples in parallel, which is a huge benefit.”

“Access to reference data is crucial, particularly for the implementation of a massively parallel targeted data analysis strategy. We worked closely with SCIEX to generate a community library of around 11,000 human proteins, plus complete libraries for yeast and tuberculosis proteomes, and some mouse data.

Some laboratories prefer to create a local library focused on their particular area of interest, while others use DIA-Umpire, a tool that analyzes SWATH data, or DIA in general, on-the-fly to build an internal spectral library. DIA-Umpire was recently compared to four other published proteomics tools, including our own OpenSWATH and the SWATH microApp in PeakView® Software from SCIEX; analyzing the same benchmarking samples with different tools delivered accurate, comparable results.

We have also developed TRIC, an alignment tool for targeted proteomics data that is a real asset for large sample cohorts; it uses a tree-building algorithm to align peptides in different samples extremely accurately, enhancing reproducibility.”

“A particularly interesting aspect of SWATH is that the data acquired can be re-mined for protein sequence variants or modifications that were not initially anticipated. For example, after acquiring data sets from a tumor biopsy, a new publication may suggest the presence of a particular fusion or genomic rearrangement. SWATH allows us to go back to the original data and re-mine it for the presence or absence of the reported product. This is a huge advantage, enabling maximum information to be derived from the data, which is particularly important for the analysis of unique and irreproducible samples, such as a tissue biopsy,” Ruedi concluded.

4 ways to learn more about SWATH:

  1. The Aebersold Group and their work
  2. TripleTOF Technology
  3. SWATH Technology
  4. How SWATH has evolved over the last 5 years

This article is from the 2017 edition of the SCIEX VISION journal. You can download the full version of the journal which includes over 10 customer research stories. Download now >

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