A multiplexed parallel reaction monitoring assay to monitor bovine pregnancy-associated glycoproteins throughout pregnancy and after gestation

Abstract

Bovine pregnancy-associated glycoproteins (boPAGs) are extensively glycosylated secretory proteins of trophoblast cells. Roughly 20 different boPAG members are known but their distribution patterns and degree of glycosylation during pregnancy are not well characterized. The objective of the present study was the development of a parallel reaction monitoring-based assay for the profiling of different boPAGs during pregnancy and after gestation. Furthermore, we investigated the effects of N-glycosylation on our analytical results. BoPAGs were purified from cotyledons of four different pregnancy stages. The assay detects 25 proteotypic peptides from 18 boPAGs in a single run. The highest abundances were found for boPAG 1 in both, glycosylated and deglycosylated samples. Strongest effects of glycosylation were detected during mid and late pregnancy as well as in afterbirth samples. Furthermore, we identified different boPAG-clusters based on the observed relative protein abundances between glycosylated and deglycosylated samples. A linkage between the impact of glycosylation and potential N-glycosylation sites or phylogenetic relation was not detected. In conclusion, the newly developed parallel reaction monitoring-based assay enables for the first time a comprehensive semi-quantitative profiling of 18 different boPAGs during pregnancy and post-partum on protein level, thereby investigating the influence of glycosylation. The results of this study provide new and important starting points to address further research on boPAGs to better understand their physiological role during pregnancy and for the development of new pregnancy detection tests.

Fig 1. Gel images of seven different purified bovine pregnancy-associated glycoproteins (boPAG) samples from four different pregnancy stages.

The protein samples (1 μg/lane) were either enzymatically deglycosylated with Peptide-N-Glycosidase F (PNGase F) (lane 4, 6, 8, 10) or left untreated (lane 3, 5, 7, 9). Molecular weights of the marker (M)-bands (lane 1, 2) are indicated on the left (kDa). Early pregnancy samples of lane 7 (b) and lane 8 (b) were not analyzed by mass spectrometry.

Fig 2. Visualization of relative protein abundances measured by Parallel Reaction Monitoring (PRM) mass spectrometry in glycosylated samples during pregnancy and post-partum.

Fig 3. Visualization of relative protein abundances measured by Parallel Reaction Monitoring (PRM) mass spectrometry in deglycosylated samples during pregnancy and post-partum.

Fig 4

Relative proportions among the different pregnancy states for (a) glycosylated samples and (b) deglycosylated samples. Note the different basic population of Total Area Fragment (a = 4,774,159; b = 7,567,626).

Fig 5. Box-and-whisker plot visualization of the percentage change of Total Area Fragment values from peptides between deglycosylated and glycosylated samples in the course of pregnancy and post-partum.

Fig 6. Overview of the results from Western blot analyses.

Box-and-whisker plot visualization represents the intensities of the fluorescence signal.

Fig 7. Mean percentage change of Total Area Fragment values from boPAGs between deglycosylated (degly) and glycosylated (gly) samples in the course of pregnancy and post-partum.