Overcoming biofluid protein complexity during targeted mass spectrometry detection and quantification of protein biomarkers by MRM cubed (MRM3)

Targeted mass spectrometry in the so-called multiple reaction monitoring mode (MRM) is certainly a promising way for the precise, accurate, and multiplexed measurement of proteins and their genetic or posttranslationally modified isoforms. MRM carried out on a low-resolution triple quadrupole instrument faces a lack of specificity when addressing the quantification of weakly concentrated proteins. In this case, extensive sample fractionation or immunoenrichment alleviates signal contamination by interferences, but in turn decreases assay performance and throughput. Recently, MRM3 was introduced as an alternative to MRM to improve the limit of quantification of weakly concentrated protein biomarkers. In the present work, we compare MRM and MRM3 modes for the detection of biomarkers in plasma and urine. Calibration curves drawn with MRM and MRM3 showed a similar range of linearity (R (2) > 0.99 for both methods) with protein concentrations above 1 mu g/mL in plasma and a few nanogram per milliliter in urine. In contrast, optimized MRM3 methods improve the limits of quantification by a factor of 2 to 4 depending on the targeted peptide. This gain arises from the additional MS3 fragmentation step, which significantly removes or decreases interfering signals within the targeted transition channels.