146 QUANTIFICATION OF PEPTIDE GROWTH FACTORS IN CATTLE UTERINE FLUID BY MULTIPLE REACTION MONITORING-LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

Abstract

Multiple reaction monitoring (MRM) allows targeted quantitative proteomics with a wide dynamic range and limit of detection down to femtomoles. We used MRM to study uterine growth factors (GF) presumed to promote embryonic development. A validated experimental model was used to recover uterine fluid (UF) and analyse GF expression in the presence or absence of embryos. Briefly, Day-6 in vitro-produced embryos (n = 50) or vehicle (sham transfer) were transferred into the uteri of each oestrus-synchronized Holstein heifer (n = 14) during nonconsecutive cycles. Blood P4 concentrations were measured on Days 0 (oestrus), 6, and 8. On Day 8, UF was recovered from embryo and sham recipients. After retrieval, UF were centrifuged and supernatants stored at –145°C. Sham and embryo UF selected for MRM were from n = 10 animals (n = 20 samples). Uterine fluid, recovered after embryo transfer, contained on average n = 43.1 ± 5.2 total and n = 34.1 ± 3.7% viable embryos per recipient. For MRM, UF samples were concentrated, and protein was precipitated and resuspended in ammonium bicarbonate. Protein (20 μg) was reduced with DTT, trypsin-digested, and desalted. Proteotypic peptides for targeted GF were selected with MRM Pilot software (ABsciex, Farmingham, MA, USA), with 3 to 5 transitions programmed for each peptide. A control, unrelated synthetic peptide was spiked as an internal standard. The area of the larger transition for the control peptide was used to normalise the area values of each other peptide. The MRM experiments were performed on a 5500 QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer (ABsciex) equipped with an Eksigent 1D+plus nanoLC chromatographic system. Data analysis was performed with Analyst 1.5.2 and MultiQuant 2.0.2 softwares (ABsciex). The area of most abundant transition for each analysed peptide was used for relative quantitation. Proteins studied were betacellulin, heparin-binding EGF-like growth factor, neuregulin, artemin, connective tissue growth factor, nerve growth factor, kit ligand, stanniocalcin-1 (STC1), early pregnancy factor (EPF), and hepatoma-derived growth factor (HDGF). Proteotypic peptides were identified in all samples for HDGF, kit ligand, STC1, and EPF (n = 1, n = 1, n = 1, and n = 3 peptides, respectively), which precluded the analysis of the remaining GF. No differences in relative abundance were detected between UF containing or not containing embryos for HDGF, kit ligand, STC1, and EPF (2.85 ± 0.6 v. 4.43 ± 0.6; 0.15 ± 0.02 v. 0.16 ± 0.02; 0.03 ± 0.00 v. 0.04 ± 0.00; and 1.20 ± 0.16 v. 1.09 ± 1.16, respectively). However, STC1 and Day 8 blood P4 were highly correlated (r = 0.71; P = 0.0004), suggesting P4 regulation of STC1. Multiple reaction monitoring-LC-MS/MS is a useful technique to identify some scarce GF in UF at different dynamic ranges.

MICINN, project AGL2012-37772 and FEDER. E. C. was supported by MEC-FPU-AP2009-5265. The authors are members of the COST Action FA1201 Epiconcept: Epigenetics and Periconception environment.