High-resolution two-dimensional electrophoresis of uterine secretion proteins using immobilized pH gradients

The protein composition of human uterine secretions was studied by high-resolution two-dimensional (2-D) electrophoresis. Secretions were collected at different phases of the menstrual cycle by sampling directly intraluminal material from 25 patients undergoing abdominal or vaginal hysterectomies. The proteins of the samples and the patients' serum were separated by isoelectric focusing, using immobilised pH gradients (IPGs, pH 4-8), followed by horizontal SDS polyacrylamide gradient gel electrophoresis (kDa 5 - 150) and silver staining. Out of a total of more than 500 spots we defined 23 spots in the protein patterns which could not be found in the patients' serum. Four spots seemed to fluctuate in relation to the cycle: the first spot (pI 5.5, kDa 31) was maximally expressed in the secretory phase, the second one (pI 7.0, kDa 31) perimenstrually in the early proliferative and late secretory phase, the third one (pi 6.4, kDa 22) in the secretory and the fourth one (pI 4.7, kDa 11) in the late proliferative to midsecretory phases. The second spot was N-terminally sequenced following micropreparative 2-D electrophoresis with narrow range IPGs. The protein could be identified as a fragment of serum albumin which was produced by cleavage between the hydrophobic amino acid leucine and arginine. It is possible that the matrix metalloproteinase MMP-7 caused the fragmentation. MMP-7 preferentially cleaves bonds of hydrophobic character, it is expressed in the endometrial glands and it can be detected in high concentrations at the beginning and the end of the menstrual cycle. Since the protein map has been obtained with a highly reproducible 2-D gel system, it will be comparable to studies from other laboratories with a high degree of reliability.