What are the iRT and HRM peptides? Which ones should I use?

What are the iRT peptides?

The iRT peptides contained in the iRT Kit are 11 non‑naturally occurring synthetic peptides which have been optimized for stability, sensitivity, and even retention time spacing over the LC gradient. The main role of the iRT peptides is to serve as internal standards to convert the retention times of your LC system into the iRT ‑indexed Retention Time‑ space. In contrast to empirical retention time, the iRT value of a peptide is stable and enables accurate prediction of peptide retention for any chromatographic setup for a given resin (e.g. C18).


What are the HRM peptides?

The HRM Calibration Kit contains the 11 iRT peptides plus an additional set of six other non‑naturally occurring synthetic peptides. The HRM Calibration Kit is needed for older versions of Spectronaut™ (7 and lower) to calibrate retention time and other parameters to enable peak detection, scoring, mass calibration, noise reduction and other steps in the data analysis process. As for Spectronaut 8, our algorithms have evolved and become more robust, and the use of HRM peptides is not required anymore.


Why should I add iRT peptides to all my samples?

Spectronaut Pulsar doesn't need the iRT peptides in data independent acquisition (DIA) runs for most types of samples. However, there are certain experiments for which spiking the iRT peptides is strongly advised. Some examples are low complexity samples, single protein digests, or IP experiments. Nevertheless, we advise to add the iRT peptides to all your runs. The cost per sample is minimal (1% or less) especially after optimizing the concentration on the newest generation of mass spectrometers.

All Biognosys workflows benefit from the use of the iRT peptides. For example, by calibrating your LC system with the iRT peptides you will increase the throughput of your targeted proteomics (multiple reaction monitoring or MRM and parallel reaction monitoring or PRM) experiments (even up to 20 times) by bundling more transitions into the run. In DIA workflows, such as hyper reaction monitoring (HRM), iRT peptides enable the generation of spectral libraries with iRT, especially for more uncommon samples.

However, these are not the only applications for the iRT peptides. Due to their stable behavior in LC‑MS setups, they are highly valuable for QC monitoring purposes. The easiest and most effective way of profiting from this feature is by using Biognosys’ quality control software solution, QuiC. However, any QC workflow benefits from the use of the iRT Kit. QuiC licenses are available in our webshop here!



Related content:

How does Spectronaut™ Pulsar use the iRT peptides?

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

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