Next generation proteomics refers to the new technologies and workflows, such as hyper reaction monitoring (HRM), multiple reaction monitoring (MRM), and parallel reaction monitoring (PRM), that are aimed at overcoming the limitations of both classical discovery proteomic approaches and traditional biochemical techniques for validation of targets. Biognosys is a leading next generation proteomics company, constantly pushing the development of technologies to provide solutions accessible to any researcher.

 

Discovery Proteomics

Discovery proteomics has traditionally relied on MS‑based shotgun workflows using data dependent acquisition (DDA). Briefly, individual precursors are picked for fragmentation in a semi‑stochastic manner, favoring the most intense peaks. As a result, a very low percentage of the detectable peptides get fragmented, and an even lower number are reliably identified (Michalski et al. 2011). This also causes a severe lack of reproducibility, even among technical replicates. Since only the selected precursors are fragmented, a large proportion of the information ‑which depends in part on the complexity of the sample‑ is lost.

The next generation proteomics alternative for discovery is data dependent acquisition (DIA). In DIA, all precursors are fragmented and MS2 data is acquired for virtually all of them. This allows to record MS1 and MS2 data from all detectable peptides in a sample and no acquirable information is lost. This feature makes DIA data significantly more convoluted than DDA data, and thus dedicated strategies for protein identification have been developed. These strategies are either peptide‑centric, mostly using spectral libraries, or spectrum‑centric, like the directDIATM workflow. One distinct advantage of DIA data is that it is a comprehensive record of a sample which can be re-processed as many times as wanted, using different libraries or targets. All these advances render DIA an excellent method for quantitative protein profiling (Bruderer et al. 2015).

 

To get the most out of DIA, Biognosys has developed an optimized DIA‑based workflow called HRM. Biognosys’ HRM technology refers to the combination of a comprehensive DIA acquisition on an high resolution mass spectrometry platform, the use of iRT spectral libraries, and data extraction using Spectronaut™ Pulsar.

HRM offers higher sensitivity, meaning more peptides are identified in a single run. The dynamic range of peptide precursor detection in a typical single shot HRM LC-MS run spans four to five orders of magnitude, with LLOQ in the range of 10s of attomole on column. In addition, it produces data of higher quality in terms of  reproducibility, data completeness, and less technical variances. HRM produces data with technical median CVs as low as 5‑10% for peptides and 5% for proteins. To know more about our technology, click here.

 

Targeted Proteomics

Validation of potential biomarker targets has been usually performed using classical antibody based techniques such as Western Blot or ELISA. However, these assays have limitation such as lack of specificity and inability to discriminate among proteoforms. In addition, these techniques can only be applied to a relatively low number of proteins at the same time.

The next generation proteomics alternative to antibody based quatification approaches is multiple reaction monitoring (MRM) and parallel reaction monitoring (PRM). These two workflows allow the quantification of hundreds of proteins in one experiment. In addition, both technologies are suitable to perform absolute quantification by using stable isotope heavy labeled reference peptides spiked into the sample at known concentrations.

 

SpectroDive is Biognosys' software solution to analyze MRM and PRM data. SpectroDive will assist you from library and panel generation and MS method setup, to data analysis and results interpretation. SpectroDive features the smartest peak picking algorithm to minimize bioinformatics resources, and a complete workflow including quality control. To get more information about targeted workflows and SpectroDive, visit our site here or read more about it here.

 

Biognosys offers ready to use assay panels for the absolute quantification of several cancer, cardiovascular, or metabolic desease biomarkers. PlasmaDeepDive and PlasmaDive multiplexed assays panels enable absolute quantification of 100 human plasma proteins in a single measurement in scheduled MRM or PRM experiments with high precision and reproducibility. Our multiplexed assays come with all the reagents, material, and protocols to assist you from a reproducible sample preparation to absolute quantification of your targets and interpretation of your results. Additional information on the targeted proteins and use cases can be found in ScienceHub section of our website here.

 

References:

Bruderer R, Bernhardt OM, Gandhi T, Miladinović SM, Cheng L-Y, Messner S, Ehrenberger T, Zanotelli V, Butscheid Y, Escher C, Vitek O, Rinner O, and Reiter L (2015) Extending the Limits of Quantitative Proteome Profiling with Data-Independent Acquisition and Application to Acetaminophen-Treated Three-Dimensional Liver Microtissues. Mol Cell Proteomics 14:1400–1410.

Michalski A, Cox J, and Mann M (2011) More than 100,000 Detectable Peptide Species Elute in Single Shotgun Proteomics Runs but the Majority is Inaccessible to Data-Dependent LC−MS/MS. J Proteome Res 10:1785–1793, American Chemical Society.

 

Related content:

Which are the Biognosys solutions for Next Generation Proteomics?

How does DIA differ from classic shotgun proteomics and DDA?

What is HRM™? Why is it better than traditional shotgun proteomics?

 

 

 

Created by SEZ. Last update 2018-03-15 by SEZ