Improving Selectivity and Sensitivity of Protein Quantitation By Lc–Hr–Ms/Ms: Determination of Somatropin in Rat Plasma

Abstract

HRMS is an interesting option for the quantitation of proteins after digestion and has the potential to improve sensitivity with minimal method development.

Intact protein quantification in biological samples by liquid chromatography – high-resolution mass spectrometry: somatropin in rat plasma

“…These results show that method performance in terms of selectivity, obtainable concentration sensitivity, accuracy and precision is essentially independent of the MEW and the number of charge states used. This is unlike what we found earlier for the signature peptides of digested somatropin [14], where interference in the chromatograms was greatly reduced and accuracy and precision were much improved by narrowing the MEW. ND: not detectable.…”

“…To support preclinical research with the 22-kDa recombinant protein somatropin in rats, we previously developed and validated an LC-HRMS method which included a digestion step and quantification of three peptides from different parts of the molecule [14]. In the present report, we describe a complementary method which enables quantification of the intact protein in rat plasma down to 10 ng/mL.…”

Effect of difluoroacetic acid and biological matrices on the development of a liquid chromatography–triple quadrupole mass spectrometry method for determination of intact growth factor proteins

“…The MRM summation of the responses of different ions has been shown to potentially improve the sensitivity of low abundance proteins and to improve the LOD [15,25,40,41]. The efficiency of these techniques are often analyte dependent and consequently, whether summation of ion intensities is beneficial needs to be determined case by case depending on the actual m / z values, extraction windows used, and the presence of matrix interferences.…”

Protein Quantification by LC–MS: A Decade of Progress Through the Pages of Bioanalysis

“…In a report published in 2012, LC-MS/MS quantification of a surrogate peptide on a Q-TOF MS was ten-times less sensitive than on a triple quadrupole [45], but a publication from 2018 showed a fourfold improvement of the achievable LLOQ on a Q-TOF compared with triple-quadrupole MS, even though the absolute instrument sensitivity of HRMS still was less favorable [46]. Analysis on a Q-TOF in the MRM mode (unit mass resolution in the quadrupole followed by high resolution in the TOF) is essential for a good signal to noise ratio: the LLOQ for an mAb in plasma obtained using TOF only without fragmentation of the surrogate peptide was found to be 25-times higher than for Q-TOF analysis in MRM mode [47].…”