PXD038465

PXD038465 is an original dataset announced via ProteomeXchange.

Title	Monitoring extended stable isotope-labeled synthetic peptides as a quality control for protease digestion in targeted, multiplexed mass spectrometry-based assays
Description	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.
HostingRepository	PanoramaPublic
AnnounceDate	2023-06-27
AnnouncementXML	Submission_2023-06-27_11:09:04.380.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Jacob Kennedy
SpeciesList	scientific name: Homo sapiens; NCBI TaxID: 9606;
ModificationList	GG; 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)
Instrument	QTRAP 6500; QTRAP 5500

Revision	Datetime	Status	ChangeLog Entry
0	2022-11-30 12:10:42	ID requested
⏵ 1	2023-06-27 11:09:04	announced

no publication

submitter keyword: MRM, immuno-affinity enrichment, quantification, human cell line, sample processing, QC

Jeff Whiteaker
contact affiliation	Fred Hutchinson Cancer Center
contact email	jwhiteak@fhcrc.org
lab head
Jacob Kennedy
contact affiliation	Fred Hutchinson Cancer Center
contact email	jkennedy@fhcrc.org
dataset submitter

Panorama Public dataset URI