Characterization of a bovine pregnancy-associated protein using two-dimensional gel electrophoresis, N-terminal sequencing and mass spectrometry.
Bovine pregnancy-associated protein (bPAP) isolated from pregnant bovine urines by two-dimensional electrophoresis (2-DE) was characterized by N-terminal sequencing, internal sequencing, and mass spectrometric analyses using matrix-assisted laser desorption/ionization-time of flight mass spectrometry and tandem mass spectrometry. Database search using the amino acid sequences and the peptide mass profiles showed that the protein is a novel bovine pregnancy-associated protein of which the N-terminus has a high similarity to collagen alpha. The protein has a molecular mass of 21 kDa and a pI of 6.1. The expression profiles of the protein from the urine of 30 pregnant and 20 nonpregnant cows characterized by 2-DE indicated that the expression of bPAP during pregnancy increased to over nmol from the pmol level basal expression of bPAP at the nonpregnant state with < 3% of false negatives and < 10% of false positives. Using the peptide sequence information, polyclonal antibodies against the bPAP protein were generated. The purified polyclonal antibodies against the peptide sequences of bPAP detected the 21 kDa protein on the blots of pregnant cow urine by Western blot analysis. In addition, analysis showed that the expression of bPAP in the urine is associated with pregnancy, but that the urine concentration of bPAP is not correlated with the duration of the pregnancy.
Pyo
Jaehoon
J
Department of Animal Science and Biotechnology, Kyungpook University, 1370 Sankyuk Dong, Buk Gu, Daegu 702-701, Korea.
Hwang
Sun-Il
SI
Oh
Jisun
J
Lee
So-Jin
SJ
Kang
Sun-Cheol
SC
Kim
Jong-Sang
JS
Lim
Jinkyu
J
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D002417:Cattle; D024042:Collagen Type I / Q000235:genetics* / Q000652:urine; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011247:Pregnancy; D011506:Proteins / Q000737:chemistry*; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000906:Antibodies; D024042:Collagen Type I; D011506:Proteins