Application of Parallel Reaction Monitoring in ¹⁵N Labeled Samples for Quantification

Accurate relative quantification is critical in proteomic studies. The incorporation of stable isotope ¹⁵N to plant-expressed proteins in vivo is a powerful tool for accurate quantification with a major advantage of reducing preparative and analytical variabilities. However, ¹⁵N labeling quantification has several challenges. Less identifications are often observed in the heavy-labeled samples because of incomplete labeling, resulting in missing values in reciprocal labeling experiments. Inaccurate quantification can happen when there is contamination from co-eluting peptides or chemical noise in the MS¹ survey scan. These drawbacks in quantification can be more pronounced in less abundant but biologically interesting proteins, which often have very few identified peptides. Here, we demonstrate the application of parallel reaction monitoring (PRM) to ¹⁵N labeled samples on a high resolution, high mass accuracy Orbitrap mass spectrometer to achieve reliable quantification even of low abundance proteins in samples.