Targeted mass spectrometry-based assays are proven to offer sensitive, selective quantification of proteins in a variety of translational applications, including cell lines, plasma/serum, dried blood spots and tissue biopsies. Recent efforts have successfully transferred targeted mass spectrometry methodologies, specifically multiplexed multiple reaction monitoring (MRM)-based assays, into the clinical environment. Yet, if MRM-based assays are to be utilized in the clinical space, quality control (QC) metrics are needed to control specifically for protease digestion, the sample processing step with the highest variability, thus ensuring optimal, reliable, and reproducible protein quantification. In this study, we evaluate the utility of monitoring extended stable isotope labeled internal standards (SIS) that are digested in situ within native protein lysates to survey the extent of sample protease digestion. Over 500 extended SIS peptides corresponding to targets of two separate multiplexed MRM assays, a direct-MRM and an immuno-MRM assay, were spiked into cell lysates which then underwent a series of systematically designed robustness experiments that stressed efficient protease digestion, including a trypsin digestion time-course as well as different protease digestion stressors. By simultaneously measuring levels of the extended and tryptic forms of the SIS peptide, the peak area ratio of extended SIS (hE) to tryptic SIS (hT), or hE/hT, was shown to effectively monitor perturbations in digestion performance. From time-course experiments, observed rates of extended SIS peptide digestion were calculated and hE/hT ratios uncovered an optimum time needed for protease digestion. When the endogenous peptides were quantified in these stressor and time-course experiments, this hE/hT QC metric reflected the sensitivity and fidelity of the MRM assays. This study demonstrates the effectiveness of monitoring extended SIS peptides within targeted, multiplexed MRM assays as a QC metric for protease digestion to ensure reliable, robust quantification of the endogenous protein targets.

In this study, we evaluated the utility of full-length extended SIS peptide measurements to provide a quality control metric for proteolytic digestion in two multiplexed MRM-based assays: an extensive direct-MRM assay targeting 398 peptides representing 246 proteins related to cancer immune function and a previously described23,32 immuno-MRM assay, which couples immunoaffinity enrichment utilizing anti-peptide antibodies to MRM-MS analysis, targeting 126 unmodified or PTM-containing (phosphorylated or ubiquitinylated) peptides representing 63 proteins related to the DNA damage response protein network. We used extended SIS peptides as internal standards for the targeted peptides in these assays and performed a series of robustness experiments systematically designed to stress or break the protease digestion. By measuring levels of both the extended and tryptic forms of the SIS (“heavy”) peptides along with tryptic endogenous (“light”), we demonstrate the ability to effectively monitor perturbations in digestion performance. These data enabled us to propose a comprehensive QC metric that ensures efficient protease digestion. Across both direct-MRM and immuno-MRM assays, we demonstrate the ability of this QC metric to generate optimal, reliable, and reproducible protein quantification, which is crucial in preclinical laboratories and an essential step toward implementing these multiplexed MRM-MS assays within the clinical environment.

Analysis of extended SIS peptide standards spiked into protein lysates from human lymphoblast cell lines (LCLs), consisting of mock (untreated) and irradiated (10Gy IR) cells, was conducted by immuno-MRM (500 ug protein, 126 peptide targets) and direct-MRM (20 ug protein, 398 peptide targets) assays. Along with a control digestion (pH 8 Tris, S/E 50, 37°C for 16 hours), two sets of robustness experiments were conducted in triplicate: a 10-time point trypsin digestion time-course from 0 minutes to 21 hours; and digestion stressors, which varied temperature (room temperature or 45°C), pH of Tris buffer (pH 6 or pH 7), protease used (no addition of Lys-C or a trypsin/Lys-C mix), or substrate-to-enzyme ratio used (S/E 25 or S/E 100). After the digestion was quenched and desalted, immuno-MRM targets were immunoaffinity enriched, and targets from both assays were analyzed separately by LC-MRM. Endogenous tryptic (“light”) and liberated tryptic SIS (“heavy” or hT), to enable quantification using peak-area-ratios (PAR) as well as full-length or ‘undigested’ extended SIS (hE) to monitor incomplete digestion were simultaneously measured in each MRM assay.