Biognosys is the leading proteomics company offering services and tools for highly multiplexed protein quantification. Its cutting-edge technology is used in biomarker research, drug and target discovery, pathway modeling, mechanisms of action studies and many other areas. Biognosys offers two distinctive technological platforms for protein analysis: discovery proteomics and targeted proteomics.
Hyper Reaction Monitoring (HRM) is a label-free discovery proteomics workflow invented at Biognosys. It allows an unmatched proteome coverage with reproducible and precise quantification of up to 9’000 proteins per sample. The HRM workflow is ideal for identifying differentially expressed proteins or highly multiplexed protein quantification on a proteome level.
The HRM workflow is based on three essential parts:
- Building a spectral library: A Spectral library is a non-redundant collection of high quality peptide assays (MS/MS spectra) that serve as a template for peptide identification during data analysis. An assay describes the characteristic features of peptide spectra such as fragment ion intensities and retention time (iRT). Spectral libraries are built from data dependent acquisition (DDA) runs on the sample type of interest.
- Acquiring data in DIA mode: Data independent acquisition (DIA a.k.a. SWATH) using high-resolution mass spectrometers enables comprehensive recording of peptide ion signatures with high resolution in mass and time. In contrast to traditional methods where single ions are isolated for further analysis, in DIA mode the mass spectrometer is programmed to cycle through broad precursor windows fragmenting multiple peptide ions together. This results in a comprehensive analysis of all detectable proteins in the sample and a high reproducibility of the results.
- Data analysis: Assay matching and protein quantification represent a big challenge in discovery proteomics. The information collected on peptides is complete but highly convoluted. By using accurate template peptide spectra from the spectral library the data first needs to be de-convoluted, before the statistical analysis and quantitation takes place. This can only be achieved using the most advanced algorithms that are also employed in our Spectronaut software.
The advantage of HRM over regular “shotgun” proteomics is that in a single measurement all detectable peptides can be quantified with high sensitivity and precision. The result of an HRM experiment is a simple and complete data matrix with precise quantity values for each protein in each sample, which can be interpreted by any researcher without the need for expert knowledge. Moreover, HRM turns a physical sample into a digital protein map which means the data can be revisited and reinterpreted at any time without the need for re-measuring the original sample.
Biognosys’ label-free targeted proteomic workflows allow absolute quantification of up to a 150 pre-defined proteins that can be analyzed in a high-through put mode in thousands of samples. In contrast to HRM where all peptides and fragment ions are recorded, targeted proteomics limits the number of peptides that will be monitored and only focuses on those peptides during acquisition to achieve the highest sensitivity and throughput for hundreds or thousands of samples. Targeted proteomics allows absolute quantification of the proteins of interest by including stable isotope standards in the analysis.
Targeted proteomics currently relies on two main approaches: Multiple and Parallel Reaction Monitoring (MRM and PRM). MRM is a well-established method for targeted proteomics and is primarily performed on triple quadrupole mass spectrometers, while its novel variant PRM was introduced recently and is performed on the latest generation of high-resolution mass spectrometric instruments.
Targeted proteomics is based on three essential parts:
- Assay development and set-up: This is a first step in targeted proteomics workflows and resembles spectral library generation in HRM. For the proteins of interest a collection of assays (also called transition or exclusion lists) is built and optimized. An assay describes the characteristic features of peptide spectra such as fragment ion intensities and retention time (iRT). These assays are later used in data acquisition.
- Data acquisition: Data acquisition is performed on triple quadrupole (MRM) or high-resolution mass spectrometric instruments (PRM). In both approaches an upfront quadrupole is used to isolate the targeted precursor ions from the assay list followed by a collision cell where product ions (transitions) are generated. While MRM only measures pre-selected transitions with the third quadrupole, PRM detects all product ions in a high resolution mass analyzer increasing the number of quantifiable proteins in one run.
- Data analysis is historically a limitation in high-throughput analysis of multiplexed MRM and PRM runs while a significant manual input is required with available tools. Biognosys overcomes these challenges with its proprietary software SpectroDive that features smartest peak picking algorithms and enables extremely fast analysis of large datasets.
Biognosys’ targeted proteomics platform is most suitable for verification or validation studies, when many samples have to be analyzed for a pre-determined set of proteins of interest.
Biognosys’ technology exploits the powerful indexed retention time (iRT) concept for achieving great results. The iRT concept allows extremely precise prediction of peptide retention time on any chromatographic system. The chromatographic retention of peptides is in principle a stable and characteristic peptide property – just like its mass. However, the actual retention time (RT) of a peptide varies between different chromatographic systems. The iRT concept makes it possible to translate the stable retention property of a peptide into accurate prediction of retention time on every possible LC set-up. This opens the path to higher multiplexing with MRM or PRM (up to 150 proteins) and improved peak picking and scoring in MRM, PRM and HRM workflows.
Biognosys recently developed a new revolutionary approach for high-precision prediction of peptide retention time (high-precision iRT) specifically designed for the targeted analysis of data independent acquisition (DIA or SWATH) in an HRM workflow. This approach extends the set of original 11 iRT peptides to thousands of anchor points derived from the actual sample and stored in Spectronaut, a professional software for DIA or SWATH data analysis. The high-precision iRT concept results in 15% more precursor IDs in a DIA analysis compared to the original iRT and 25% more precursor IDs in a DIA run when retention time prediction was not used for spectral library generation (Bruderer et al. 2016).