NEW YORK (GenomeWeb) – Researchers at the University of Washington have applied parallel-reaction monitoring (PRM) mass spec to the development of targeted phosphoproteomics assays.
The method, which was detailed in a paper published this week in Nature Methods, leverages PRM's reproducibility and simplicity to generate what the authors, led by UW researcher Judit Villén, described as rapid "plug and play" targeted phosphoprotein assays.
PRM is essentially a variety of data-independent mass spec in which the mass spectrometer, rather than analyzing the full range of a sample, is trained on a more targeted mass and time window. Several years ago, researchers including Bruno Domon, head of the Luxembourg Clinical Proteomics Center, and University of Wisconsin-Madison's Josh Coon began exploring PRM on high-resolution instruments like Thermo Fisher Scientific's Q Exactive as an alternative to traditional triple quadrupole-based targeted protein quantitation.
Such an approach has various potential advantages. For instance, because their analyzers are able to collect data on a wide range of ions, high-resolution machines could allow for easier assay development and better specificity.
In a triple quad-based SRM assay, the first quadrupole isolates a target precursor ion, which is then fragmented in the second quadrupole, after which a set of preselected product ions are detected in the third quadrupole. By contrast, PRM approaches use the upfront quadrupole of a Q-TOF or Q Exactive machine to isolate a target precursor ion, but then monitor not just a few but all of the resulting product ions.
Because of this, researchers don't have to determine upfront what the best transitions to monitor will be, significantly reducing assay development time.
The larger number of product ions monitored via PRM should also improve the specificity of the analysis, since more transitions will be available to confirm a peptide ID. This might also reduce the effects of co-isolating background peptides.
As the Nature Methods authors noted, developing targeted assays for phosphoproteins is particularly challenging as phosphorylation changes local charge distributions and interferes with digestion, complicating the process of selecting the best peptide sequence and charge state to monitor in a targeted assay.
In developing their PRM assays for phosphoproteomic work, the researchers compiled a large scale LC-MS/MS database of phosphopeptide sequences, consisting of around 1,000 mass spec runs containing more than 7.5 million phosphopeptide spectral matches and 109,611 phosphorylation sites on 11,428 proteins.
They used this database to analyze the effectiveness of conventional data dependent acquisition mass spec for phosphoproteomics, finding, for instance, that deep fractionation was required to see a large percentage of phosphopeptides, and that reproducibility across runs was poor even in experiments where deep fractionation was used. For instance, in such experiments, only 8 percent of phosphopeptides were observable in at least 50 percent of runs.
They also examined the charge and cleavage state specificity of the phosphopeptides, finding that most sites were observed in just one cleavage state and had "moderately specific" charge states.
Selecting 101 phosphopeptides, they developed a one-hour PRM assay for measuring them, analyzing their samples using PRM, DIA, and DDA approaches and finding that PRM outperformed DIA and DDA in terms of the number of peptides detected and their reproducibility.
They then applied the same approach to quantifying phosphorylation sites on proteins in the IGF-1/AKT signaling pathway in MCF7 cells before and after stimulation with IGF-1. Notably, their PRM assay was able to reproducibly quantify phosphorylated AKT isoforms that, the authors said, are indistinguishable by antibody-based approaches and difficult to detect using DDA mass spec, even with extensive fractionation.
The study, the researchers wrote, demonstrated "the potential of label-free PRM assays for robust, high-throughput, targeted phosphoproteome analysis." They have established a web-based tool that allows other researchers to build their own PRM assays by querying their database for parameters like peptide selection and retention time scheduling.