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 of the amniotic fluid of patients in preterm labor.

Study design: Amniotic fluid was obtained by amniocentesis 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 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 (> or =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. Enzyme-linked immunosorbent assays (ELISA) as well as surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS)-based on-chip antibody capture immunoassays 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 IAI. (3) Proteins that 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 that were over-expressed in group 3 included fibrinopeptide B, transferrin, major histocompatibility complex (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). (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 the amniotic fluid of patients with preterm labor. 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.

Figure 1

ProteoVue Maps of the three pools of amniotic fluid after treatment with reducing agents. 1a) patients with preterm labor without infection/inflammation who delivered at term (group 1); 1b) patients who delivered preterm without intra-amniotic infection/inflammation (group 2); 1c) patients who delivered preterm with intra-amniotic infection/inflammation (group 3).

Figure 2

ProteoVue Maps of the two pools of untreated amniotic fluid in the second set of experiments. (a) Group 1: patients with preterm labor without infection/inflammation who delivered at term (b)Group 3: patients who delivered preterm with intra-amniotic infection/inflammation. The region indicated by the box in (a) contains the fractions from which IGFBP-1 was subsequently identified.

Figure 3

DeltaVue map for untreated amniotic fluid, overlaying the protein concentration of patients with preterm labor without infection/inflammation who delivered at term (group1 - in red) and patients who delivered preterm with intra-amniotic infection/inflammation (group 3 - in green). The center image is the difference map for proteins, with the colored bands indicating protein over-expression from the respective sample. The green and red chromatograms (to the left and right of the center image, respectively) are the protein profiles for the pI 4.9 – 5.2 CF fraction of each group. The arrow and the square indicate one fraction that was collected for identification of a protein that was over expressed in group 1 compared to group 3.

Figure 4

One dimensional SDS-Page for untreated amniotic fluid, comparing several fractions of patients with preterm labor without infection/inflammation who delivered at term (group1) and patients who delivered preterm with intra-amniotic infection/inflammation (group 3). In the third fraction analyzed (identified as 1.4.1 and 2.4.1 and corresponding to the RT fractions with an over expression of proteins of group 1 into the PI 4.9-5.2 CF fractions). Two spots at an approximate molecular weight of 30 Kda and 60 Kda were identified in group 1 but not in group 3. Those two spots were excised and IGFBP-1 was subsequently identified by LC-MS/MS in both. No protein was identified in the two corresponding spots of group 3.

Figure 5

ProteoVue Maps of the two pools of reducing solubilization buffer-treated retentates from concentrated amniotic fluid (experiment 3). (a) Patients with preterm labor without infection/inflammation who delivered at term (group 1).(b) Patients who delivered preterm with intra-amniotic infection/inflammation (group 3).

Figure 6

DeltaVue map overlaying the proteins from the pI 5.5-5.8 CF fractions of patients with preterm labor without intra-amniotic infection/inflammation who delivered at term (group 1 - in red) and patients who delivered preterm with intra-amniotic infection/inflammation (group 3 - in green) according to the hydrophobicity. The center image is the difference map of proteins concentrations between the two groups. The arrows indicate two fractions in which proteins were over expressed in group 3, identified as Down Syndrome Critical Region Protein 2 and human peptide 8 (HP8).

Figure 7

Amniotic Fluid IGFBP-1 concentration determined by ELISA in the three groups of patients with preterm labor. No significant differences in the mean concentrations of IGFBP-1 among the three groups of patients were detected.

Figure 8

SELDI-TOF MS ProteinChip immunoassays of amniotic fluid. (a) The IgG negative control for group 1 (preterm labor without infection/inflammation who deliver at term) (b) The IGFBP-1 capture antibody assay for the same amniotic fluid. (c) The IgG negative control for group 3 (preterm labor with intra-amniotic infection/inflammation). (d) The IGFBP-1 capture antibody assay for the same amniotic fluid (group 3). Peaks near 13 kDa correspond to proteolytic fragments of IGFBP-1, while the peak near 25 kDa correspond to the intact IGFBP-1 protein. Comparison of Figures (b) and (d) reveals evidence for proteolytic cleavage occurring in amniotic fluid from women with intra-amniotic infection/inflammation (group 3), but not in those without it (group 1).