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PXD038465 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleMonitoring extended stable isotope-labeled synthetic peptides as a quality control for protease digestion in targeted, multiplexed mass spectrometry-based assays
DescriptionTargeted 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.
ReviewLevelNon peer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportSupported dataset by repository
PrimarySubmitterJacob Kennedy
SpeciesList scientific name: Homo sapiens; NCBI TaxID: 9606;
ModificationListGG; Phospho; Oxidation; Carbamidomethyl; Label:13C(3)15N(1); Label:13C(6)15N(1); Label:13C(6)15N(2); Label:13C(9)15N(1); Label:13C(6)15N(4)
InstrumentQTRAP 6500; QTRAP 5500
Dataset History
RevisionDatetimeStatusChangeLog Entry
02022-11-30 12:10:42ID requested
12023-06-27 11:09:04announced
Publication List
no publication
Keyword List
submitter keyword: MRM, immuno-affinity enrichment, quantification, human cell line, sample processing, QC
Contact List
Jeff Whiteaker
contact affiliationFred Hutchinson Cancer Center
contact emailjwhiteak@fhcrc.org
lab head
Jacob Kennedy
contact affiliationFred Hutchinson Cancer Center
contact emailjkennedy@fhcrc.org
dataset submitter
Full Dataset Link List
Panorama Public dataset URI