Mol Endocrinol. microorganisms. Intra-amniotic inflammation was defined as an elevated amniotic fluid interleukin (IL)-6 (2.3 ng/mL). Two-dimensional (2D) chromatography was utilized for analysis. The first Difopein dimensions 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 made up of 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 utilized for confirmation of a specific protein Difopein 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 Difopein not switch, 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 crucial 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 including 2D chromatography, mass spectrometry, and immunoassays allows identification of proteins that are differentially regulated in amniotic fluid of patients with preterm labor. Akt1s1 Specifically, the amount of the IGFBP-1 fragments at approximately 13.5 kDa was found to be increased in patients with IAI, while the amount of the intact form of IGFBP-1 was decreased. fluid cultures. Amniotic fluid white blood cell (WBC) count, red blood cell (RBC) count and glucose concentrations were performed. The results of these assessments were utilized for subsequent clinical management. Patients with amniotic fluid RBC count of more than 100 cells were excluded. Intra-amniotic inflammation was defined as amniotic fluid concentration of interleukin-6 (IL-6) higher than 2.3 ng/ml. This cut-off of IL-6 concentration was derived from applying a receiver-operating characteristic curve to amniotic fluid concentrations of IL-6 of patients with spontaneous preterm labor included in this study for the identification of Difopein patients who delivered within 7 days of amniocentesis. Many of these samples have been used previously in several studies focusing on term and preterm parturition, cytokines, chemokines, arachidonic acid metabolites, and other biological markers of disease. Amniotic fluid preparations for proteomics studies Three different methods for preparing amniotic fluid samples for proteomic investigation via 2D separation chromatography were investigated. We found that these methods experienced a profound impact on the ability to detect, characterize and quantify differential protein expression in amniotic fluid, and describe here each method and its strengths and limitations. For details of amniotic fluid preparations and analytical methods, the reader is usually referred to the Appendix section. A brief description of the methods is provided as follows: Reduced amniotic fluid In a first set of experiments, amniotic fluid samples were treated with reducing brokers to improve solubilization. This method was successful only in identifying high-abundance proteins. Untreated amniotic fluid In a second set of experiments, untreated amniotic fluid was buffer-exchanged and directly injected into the chromatofocussing column. Concentrated amniotic fluid In a third set of experiments, protein from large volumes of amniotic fluid from groups 1 and 3 was concentrated by centrifugation with a molecular excess weight cutoff filter (30 kDa). The retentate was treated with a reducing.