Objective
Simultaneous analysis of the protein composition of biological fluids is now possible. Such an approach can be used to identify biological markers of disease and to understand the pathophysiology of disorders that have eluded classification, diagnosis, and treatment. The purpose of this study was to analyze the differences in protein composition in amniotic fluid of patients in preterm labor.
Study Design
Amniotic fluid was obtained by amniocenteses from three groups of women with preterm labor and intact membranes: (1) women without intra-amniotic infection/inflammation (IAI) who delivered at term; (2) women without intra-amniotic IAI who delivered a preterm neonate; and (3) women with IAI. Intra-amniotic infection was defined as a positive amniotic fluid culture for microorganisms. Intra-amniotic inflammation was defined as an elevated amniotic fluid interleukin (IL)-6 (≥2.3 ng/mL). Two-dimensional (2D) chromatography was used for analysis. The first dimension separated proteins by isoelectric point, while the second, by the degree of hydrophobicity. 2D protein maps were generated using different experimental conditions (reducing agents as well as protein concentration). The maps were used to discern subsets of isoelectric point/hydrophobicity containing differentially expressed proteins. Protein identification of differentially expressed fractions was conducted with mass spectrometry. ELISA immunoassays as well as surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS)--based on-chip antibody capture immunoassay were also used for confirmation of a specific protein that was differentially expressed.
Results
1) Amniotic fluid protein composition can be analyzed using a combination of 2D liquid chromatography and mass spectrometry for the identification of proteins differentially expressed in patients in preterm labor; 2) While total insulin-like growth factor-binding protein-1 (IGFBP-1) concentration did not change, IGFBP-1 fragments at about 13.5 kDa were present in patients with intra-amniotic IAI; 3) proteins which were over-expressed in group 1 included Von Ebner gland protein precursor, IL-7 precursor, apolipoprotein A1, tropomyosin sk1 (TPMsk1) fragment, ribosomal protein S6 kinase alpha-3 and alpha-1-microglobulin/bikunin precursor (AMBP); 4) proteins which were over-expressed in group 3 included fibrinopeptide B, transferrin, (MHC) class 1 chain-related A antigen fragment, transcription elongation factor A, sex-determining region Y (SRY) box 5 protein, Down syndrome critical region 2 protein (DSCR2), and human peptide 8 (HP8); and 5) one protein, retinol binding protein, was over-expressed in women who delivered preterm, regardless of the presence of IAI.
Conclusions
A combination of techniques involving 2D chromatography, mass spectrometry, and immunoassays allows identification of proteins that are differentially regulated in amniotic fluid of patients with preterm labor. Specifically, the amount of the IGFBP-1 fragments at approximately 13.5 kDa ...