Reproducibility in research is key, however it is challenging for many techniques across multiple disciplines. A survey1 published in Nature in 2016 showed that more than 70 % of researchers were not able to reproduce another scientist's experiments, with more than half unable to reproduce their own experiments. A recent study2 looked to address the quantitative and qualitative reproducibility of SWATH-mass spectrometry (MS), a data independent acquisition technique which allows a complete and permanent recording of all fragment ions of the detectable peptide precursors present in a biological sample. “Based on the work that was already coming out at the time it was clear that SWATH Acquisition was going to be an important quantitative technique for proteomics, and labs were already demonstrating that large numbers of proteins could be quantified in proteomic samples very reproducibly on smaller studies,” commented Dr Christie Hunter, one of the authors of the study that was published in Nature Communications. Christie went on to say that “there had also been a number of other important multi-site studies that had been done for other proteomic techniques, which had demonstrated the importance of testing a technique in that manner”.
Targeted proteomics, incorporating MS, has been a powerful research tool offering reproducible results for some time. However, the technique is limited in the number of proteins that may be detected in a sample. SWATH-MS does not have this limitation, making it a candidate technique for large scale proteomic projects. Previous SWATH-MS studies have demonstrated high intra-lab reproducibility, but inter-lab variation had not been assessed.
“We had two questions that we thought were important to test at this point in time. Can multiple labs generate very similar data on the same sample? Does SWATH Acquisition enable the generation of large scale datasets across labs to enable large scale quantitative biology experiments?” commented Christie. In this study, 11 laboratories across the globe analyzed standard samples, consisting of 30 stable isotope-labelled synthetic peptides, that had been diluted in a complex protein background of digested HEK293 cells at concentrations that spanned six orders of dynamic range. The five samples were analyzed multiple times on the same day, on multiple days and across multiple sites. In total, 229 data files were generated by the participating labs which were then compared centrally for quantitative and qualitative concordance.
Results showed that >4000 proteins from HEK293 cells could be consistently detected and reproducibly quantified. Detection of the peptide standards demonstrated that the sensitivity, dynamic range and reproducibility established with SWATH-Acquisition are uniformly achieved.
This study demonstrated that detection of large numbers of proteins from highly heterogeneous samples is not only feasible but provides reliable and reproducible quantitative results. Comparative quantitative analysis of large numbers of proteomes is becoming more accessible and it is therefore expected that research where this capability is a prerequisite is increasing. Applications where this could have an impact include analyses of clinical material from large patient cohorts (e.g., biomarkers, personalized medicine research), association of protein abundances to genomic features using genetic reference collections or wild-type populations (e.g., quantitative trail locus or genome wide association studies), or large-scale perturbation screens using in vitro model systems (e.g., drug screens), are now feasible.
The data demonstrates a significant advance in the robustness of large-scale data acquisition in quantitative proteomics, and the paper2 concludes that the results are expected to increase confidence in SWATH-MS. “With the increasing focus on precision medicine research and a much better understanding of the need to analyze more samples, the ability to profile large numbers of samples quantitatively will be critical,” said Christie.
1. Baker M. 1,500 scientists lift the lid on reproducibility. Nature. 2016 May 26;533(7604):452-4. doi: 10.1038/533452a. PubMed PMID: 27225100.
2. Collins BC, Hunter CL, Liu Y, Schilling B, Rosenberger G, Bader SL, Chan DW, Gibson BW, Gingras AC, Held JM, Hirayama-Kurogi M, Hou G, Krisp C, Larsen B, Lin L, Liu S, Molloy MP, Moritz RL, Ohtsuki S, Schlapbach R, Selevsek N, Thomas SN, Tzeng SC, Zhang H, Aebersold R. Multi-laboratory assessment of reproducibility, qualitative and quantitative performance of SWATH-mass spectrometry. Nat Commun. 2017 Aug 21;8(1):291. doi: 10.1038/s41467-017-00249-5. PubMed PMID: 28827567; PubMed Central PMCID: PMC5566333.