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ISB and ETH Team Completes SRMAtlas Targeted Proteomics Resource

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NEW YORK (GenomeWeb) – A team led by researchers at the Institute for Systems Biology and the Swiss Federal Institute of Technology Zurich have completed the Human SRMAtlas, a collection of selected reaction-monitoring mass spectrometry assays to the human proteome.

Detailed in a paper published last month in Cell, the resource contains data on 166,174 proteotypic peptides and allows for quantification of 20,123 of the 20,203 annotated human proteins in the UniProtKB/Swiss-Prot database. It also contains several thousand assays to protein isoforms and post-translational modifications, Robert Moritz, director of proteomics at ISB and one of the leaders of the project, told GenomeWeb.

Launched in 2009 with $2.7 million in funds from the American Recovery and Reinvestment Act as well as $4.1 million from the European Research Council, the SMRAtlas is intended as a resource to aid researchers interested in using targeted mass spec for protein quantitation. While uptake of targeted mass spec methods like SRM has grown quickly in recent years, it remains something of a tool for specialists, and developing assays from scratch can be a time-consuming process. The SRMAtlas aims to simplify that process by providing ready-made assays to the full complement of the human proteome.

In a sense, it is part of a larger goal within the targeted proteomics community to shift protein research within the life sciences away from antibody-based techniques toward mass spec methods. While antibodies remain the tools of choice for most life science researchers, high-quality antibodies exist for only a relatively small number of proteins, which, mass spec proponents argue, constrains research, with scientists tending to focus their efforts on molecules that can be effectively measured using antibodies.

Indeed, the Human Proteome Organization has formed working groups of researchers and vendor representatives to explore the feasibility of mass spec-based instruments that would allow non-mass spectrometrists to run targeted quantitation experiments in much the way automated immunoassay platforms do today.

The SRMAtlas doesn't go so far as to make targeted mass spec accessible to complete novices, Moritz noted, but he said he and his colleagues have found it effective in spreading usage of the technique within mass spectrometry circles.

"For the more inexperienced people, this is a perfect way for them to get into the technology," he said, noting that despite increasing use of targeted quantitation in proteomics, much of the field remains focused on shotgun-style experiments.

The SRMAtlas is also designed to be compatible with more recently developed high-resolution targeted mass spec techniques like parallel reaction-monitoring and Swath-style data-independent acquisition methods, Moritz said, noting that all of the data was built using both low-resolution triple quadrupole instruments and high-resolution QTOFs.

When the SRMAtlas project launched in 2009, methods like Swath and PRM were still under development, but since then both have become increasingly popular techniques. In a 2010 interview with GenomeWeb discussing development of the SRMAtlas, Moritz highlighted the decision to collect data for the resource on high-resolution instruments.

"This future-proofs the data," he said. "It extends normal quadrupole technology, and as [vendors] manufacture [new] high-resolution techniques, this data will be immediately applicable to that type of technology because we already have it in very high resolution."

Developing an SRM assay involves identifying proteotypic peptides (tryptic peptides specific to a particular protein), then determining which of those peptides are most amenable to quantitation via mass spec as well as the best conditions and settings for quantitating them, and then determining what precursor and product ion pairs, or transitions, are best for obtaining good quantitation.

The SRMAtlas provides this information, eliminating a considerable portion of assay development work. However, as Moritz noted, when using the assays in actual samples, their effectiveness can vary depending on what interferences are present in those samples. For instance, he said, an analyte present in plasma might interfere with a particular transition for one SRM assay, meaning researchers should use a different one. That same transition might work fine, though, in urine, while interferences in that sample source could limit the effectiveness of a different transition.

"I think it really comes down to a transition-by-transition basis depending on the sample," Moritz said.

To help mitigate this issue and to enable greater specificity in protein quantitation, the researchers developed assays to multiple peptides per protein. Of the 20,123 proteins addressed by the atlas, 17,779 are targeted by assays to five or more peptides; 1,242 by assays to four peptides; 875 by assays to three peptides; 570 by assays to two peptides; and 348 by assays to one peptide.

Moritz said that he and his colleagues hope to continue adding peptides in future versions of the atlas to further flesh out the targeted proteins.

"As we go on, we will be filling them out with more peptides to go for the whole complement of every protein rather than the most proteotypic," he said.

The researchers will also continue to add assays as new proteins are identified through efforts like the Human Proteome Project, although Moritz said that much of his team's earlier work, which used peptides predicted by the underlying genetic sequence for proteins that had not yet been experimentally observed, has held up well.

"When we first started, the community really only knew about 50 percent of the proteome," he said, which necessitated extensive peptide predictions.

Since then, advances have enable researchers to identify around 75 percent of the proteome. "And we have found that our predictions were good enough that 85 percent of [them] held true with the new identifications being made," Moritz said.

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