Method Development in Mass Spectrometry-based Proteomics for Determination of Early Pregnancy in Dogs

Abstract: This project is concerned with method development in mass spectrometry (MS)-based proteomics in order to find putative biomarkers for early pregnancy ofdomesticated dogs. It is of importance for dog breeders to know whether the dogsbecome pregnant post-mating. Unlike humans, dogs are not known to possess aspecific hormone that indicates fetal development; therefore other biomarkers mustbe investigated. The approach of choice in this project was to look at proteins throughMS-based proteomics. For this purpose, serum samples from 11 pregnant dogs (case,different breeds) and 7 non-pregnant dogs (control, all beagle dogs) were sampledbefore-hand at the Swedish University of Agricultural Sciences. Each dog wassampled Day 1, Day 8, Day 15, Day 22 and Day 29 after optimal mating. Twodifferent proteomics approaches were conducted: Bottom-up (“Shotgun”) proteomicsand targeted proteomics (“targeted analysis”). In this study, Label-free Quantification(LFQ) was employed, which is a relative quantitative technique. The massspectrometer of choice was the Quadrupole-Orbitrap QExactive plus massspectrometer coupled to a nano-Ultra Performance Liquid Chromatography (UPLC).Method optimization was done with respect to concentration of samples prior to MSanalysis, as well as different LC-gradients. From shotgun screening experiments, itwas possible to identify 252 proteins. Ultimately, 9 proteins were investigated usingtargeted final analysis: CRP, SERPINC1, CP, PROS1, SERPING1, A2M, AGP,SERPINA1 and HP. For targeted final analysis, 21 peptides were considered.Calibration curves were constructed using 8 of the 21 targeted peptides; 1 peptide perprotein, except for HP which had 2 peptides per protein. The SERPINA1 and CPproteins had no appropriate peptides for targeted final analysis and were thusexcluded. It was confirmed that CRP was up-regulated in case dogs compared tocontrol dogs. The other investigatedproteins showed no significant signs of regulation. In order to improve the results; itwould be desirable to include more dogs in the study which would benefit thestatistics of protein regulation. However, the use of isotopic labeled standards andemployment of a Parallel Reaction Monitoring (PRM) method should be prioritizedfor obtaining absolute quantitative data.