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Multiple reaction monitoring assays for large-scale quantitation of proteins from 20 mouse organs and tissues

ABSTRACT: We developed 7,184 quantitative multiple reaction monitoring mass spectrometry (MRM-MS) assays that measure 2,118 unique proteins in 20 mouse organs and tissues to enable system-level analysis of protein expression in mice. Targets for assay development were selected by untargeted MS analysis, and corresponding stable isotope-labelled standards were synthesized in-house. Development experiments determined the lower limit of quantitation, linear range, and repeatability of each assay. Subsequently, the developed assays were formatted into panels tailored to each organ or tissue type and were used to measure normal protein concentrations in samples from three mouse strains: C57BL/6NCrl, NOD/SCID, and BALB/cAnNCrl. In total 5,149 measurements were obtained corresponding to 1,691 proteins, and results included sex- and strain-specific profiles in protein abundances. Collectively these assays are the largest group of quantitative MRM-MS assays established and implemented in mice to date, and enable accurate quantitation of mouse proteins for molecular phenotyping. Furthermore, the measured concentration data provide important biological information for experimental design involving mouse models.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Heart, Testis, Spleen, Brain, Skeletal Muscle, Blood Plasma, Brown Adipose Tissue, Lung, Liver, Salivary Gland, Kidney, Eye, Erythrocyte, Ovary, Epididymis, White Adipose Tissue, Skin, Seminal Vesicle, Pancreas

SUBMITTER: Sarah Michaud

LAB HEAD: Sarah Michaud

PROVIDER: PXD021333 | Pride | 2024-01-03

REPOSITORIES: Pride

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Project description:The current state of proteomics requires a choice between targeted and discovery methods. The former provides exceptional quantitation and data completeness, but only with few analytes. Discovery proteomics can identify hundreds and thousands of proteins in a sample, but with poor quantitation and data completeness. We have established and optimized a method that combines targeted and data-independent acquisition for absolute quantitation of all plasma proteins in a single sequential window acquisition of all theoretical fragment ions (SWATH) acquisition run using a panel of spike-in standards (SIS). We compared the absolute quantitation (AQ) of SWATH and high-resolution multiple-reaction monitoring (MRM-HR) acquisition methods using the 100 protein PlasmaDive SIS panel spiked into human plasma. SWATH provided equivalent quantitation and differentially abundant protein profiles as MRM-HR. Absolute quantities of the SIS peptides from the SWATH data were used to estimate the absolute quantities (eAQ) for all the proteins in the run. The eAQ values provided similar quantitation and differentially abundant protein profiles as AQ and protein group (PG) values, and the eAQ method was the only scheme where all selected proteins were verified by MRM-HR. We applied the eAQ method to a cohort of 16 non-small cell lung cancer (NSCLC) patients receiving immunotherapeutics and found that fibronectin (FN1) and proteoglycan 4 (PRG4) were useful markers for predicting patients who would stay on these agents for at least 6 months (area under the curve of 0.8438 and 0.6735, respectively). Thirteen additional plasma samples confirmed that FN1 and PRG4 are putative biomarkers (positive predictive value is 100% and 85.7%, respectively) for a prolonged (>6 months) response to immunotherapeutic agents. In conclusion, we describe an optimized method for absolute quantification of all proteins in a SWATH run, and this approach is amenable for the identification of plasma biomarkers.

			Action	DRS
	Brain_Female.raw	Raw
	Brain_Male.raw	Raw
	Brain_Male_Female.msf	Msf
	Brain_Male_Female.msfView	Msf
	Brain_Male_Female.pdResult	Other

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Multiple reaction monitoring assays for large-scale quantitation of proteins from 20 mouse organs and tissues

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