Molecular phenotyping of laboratory mouse strains using 500 multiple reaction monitoring mass spectrometry plasma assays

Abstract

Mouse is the predominant experimental model for the study of human disease due, in part, to phylogenetic relationship, ease of breeding, and the availability of molecular tools for genetic manipulation. Advances in genome-editing methodologies, such as CRISPR-Cas9, enable the rapid production of new transgenic mouse strains, necessitating complementary high-throughput and systematic phenotyping technologies. In contrast to traditional protein phenotyping techniques, multiple reaction monitoring (MRM) mass spectrometry can be highly multiplexed without forgoing specificity or quantitative precision. Here we present MRM assays for the quantitation of 500 proteins and subsequently determine reference concentration values for plasma proteins across five laboratory mouse strains that are typically used in biomedical research, revealing inter-strain and intra-strain phenotypic differences. These 500 MRM assays will have a broad range of research applications including high-throughput phenotypic validation of novel transgenic mice, identification of candidate biomarkers, and general research applications requiring multiplexed and precise protein quantification.

Concentration Determination of >200 Proteins in Dried Blood Spots for Biomarker Discovery and Validation

“…After preanalytical sample preparation (described above) of two replicate DBS samples, the samples were subjected to solid phase extraction (25) and lyophilisation. Samples were solubilized in 210 l of 3% aqueous acetonitrile in water and used for LC-MS/MS on an Orbitrap Fusion Tribrid coupled to an EASYnLC 1000 HPLC system via a Nanospray Flex NG source (Thermo Fisher Scientific), as previously described (26,27). The only modification made was to inject 1 l of each technical replicate, which was equivalent to ϳ 1 g of total protein.…”

“…Raw files were created using XCalibur 3.0.63 (Thermo Scientific) software and analyzed with Thermo Proteome Discoverer 2.2.0.388 software suite (Thermo Scientific). The raw data files were processed as previously described (26) with the following modifications: the peak lists were submitted to Mascot 2.4.1 and Sequest servers using the UniProt human database (28) which contained 20,338 protein sequences. The enzyme was set to Trypsin (Full) with max missed cleavage set to 2.…”

“…Response Curve-CPTAC experiment 1 (20) was used as a guide to assess the lower limit of quantification (LLOQ) for each target peptide. Using SIS peptides to spike trypsin digested DBS (DBS matrix), the reverse-curve (26,27) approach was used to generate an eight-point response curve (in addition to a blank), to determine the LLOQ. The 336 SIS peptides to be tested were used to make working solutions of SIS peptides containing 5000 fmol/l of peptide in 3% ACN/H 2 O.…”

“…Multiday Validation of Assay Analytical Repeatability-For peptides for which an LLOQ was determined, based on the criteria defined above, a multi-day assessment of repeatability was performed, using CPTAC experiment 2 (20) as a guide. Repeatability was assessed using the reverse-curve approach (26,27), which involves varying the SIS concentration and using NAT and/or endogenous proteolytic peptides as normalizers, or by using the SIS peptide without normalization, for targets for which the NAT peptide was not available or where the endogenous proteolytic peptide was contributing to the variability. DBS matrix spiked with NAT peptides was used for the blank data points which would later be used to assess the limit of detection, defined as three standard deviations higher than the signal measured across the blank samples (three replicates).…”

“…Measurement of Lower Limit of Quantification and Linear Range-The LLOQ and the range of linearity for the SIS peptides were determined in a DBS matrix based on sevenpoint response curves using the reverse-curve strategy (26,41), i.e. the concentration of stable isotope labeled standard (SIS) peptides is varied and the corresponding unlabeled standard (NAT) and/or endogenous proteolytic peptides are used as normalizers.…”