Proteomic profiling of amniotic fluid in preterm labor using two-dimensional liquid separation and mass spectrometry

Abstract

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 ...

Low abundance plasma proteins in labour

“…IGFBP1 was also increased in both emergency caesarean section groups, and its expression is regulated by cytokines (IL1b) and hCG through HNF1A (Tang & Gurpide 1993). Epidemiological studies have shown that elevated maternal serum IGFBP1 levels in the second and early third trimesters significantly associate with preeclampsia, intrauterine fetal growth restriction and other pregnancy complications (Bujold et al 2008). At term, IGFBP1 levels are higher in spontaneous labour than in elective caesarean section; this may reflect the higher incidence of fetal hypoxia and/or fetal distress in active labour (Hills et al 1996) and is supported by the results of elevated IGFBP1 in both ILEC and SLEC, as a possible consequence of fetal distress (Marchini et al 2005).…”

“…AMBP is reported to be increased in the amniotic fluid of women delivering at term compared with preterm deliveries (Bujold et al 2008). Further studies are required to understand the involvement of this group of proteins in pregnancy and parturition.…”

Proteomics Approaches in Pre-Eclampsia Research

“…Some of the most exciting developments thus far have been in the investigation of preterm labour, [36][37][38][39][40] however this review will focus on the application of these techniques to pre-eclampsia. To date, several different proteomic techniques have been applied to cultured cytotrophoblasts cells, primary placental tissue and bodily fluids such as plasma, urine, CSF and amniotic fluid.…”

Comment and reply on: Metabolomics of amniotic fluid and preterm delivery

“…High-dimensional biology techniques are not generally designed to be rapid methods, but rather, to be comprehensive in obtaining the best possible description of the composition of a biological fluid. However, once a biomarker (or a set of biomarkers) is identified using discovery platforms, targeted assays can be developed to detect such specific differences [5,6,10]. The purpose of our study and article was to demonstrate that metabolomics allows the detection of differences in the composition of women in the three different clinical groups.…”

“…serum or plasma, urine, cerebrospinal fluid, amniotic fluid, tissues, etc.) among patients, typically between healthy individuals and those with disease [4][5][6][7]. However, highdimensional biology techniques could also be used to classify patients with a particular clinical entity into those with good and bad prognosis [8], or even uncover the degree of heterogeneity of a particular entity [9].…”