Comprehensive proteomic analysis of human cervical-vaginal fluid.

Cervical-vaginal fluid (CVF) is a potential rich source of biomarkers for enhancing our understanding of human parturition and pathologic conditions affecting pregnancy. In this study, we performed a comprehensive survey of the CVF proteome in pregnancy utilizing multidimensional liquid chromatography (2D-LC) coupled with mass spectrometry and gel-electrophoresis-based protein separation and identification. In total, 150 unique proteins were identified using multiple protein identification algorithms. Metabolism (32%) and immune response-related (22%) proteins are the major functional categories represented in the CVF proteome. A comparison of the CVF, serum, and amniotic fluid proteomes showed that 77 proteins are unique to CVF, while 56 and 17 CVF proteins also occur in serum and amniotic fluid, respectively. This data set provides a foundation for evaluation of these proteins as potential CVF biomarkers for noninvasive diagnosis of pregnancy-related disorders.

Identification of novel protein biomarkers of preterm birth in human cervical-vaginal fluid.

Spontaneous preterm birth (SPTB) is a major contributor to perinatal morbidity and mortality. However, the diagnosis of preterm labor (PTL) that leads to preterm birth is difficult, and there is a pressing need for improved diagnosis. We utilized multidimensional liquid chromatography-tandem mass spectrometry (LC/LC-MS/MS; MudPIT) and Fluorescence two-dimensional differential in-gel electrophoresis (2D-DIGE) to identify potential biomarkers of PTL and SPTB. MudPIT analysis identified 205 proteins in cervical-vaginal fluid (CVF), 28 of which exhibited significant differences in pairwise and progressive comparisons. Calgranulins, annexins, S100 calcium-binding protein A7, and epidermal fatty acid binding protein were abundant in CVF and differentially present in PTL and SPTB samples, as were the serum proteins alpha-1-antitrypsin, alpha1-acid glycoprotein, haptoglobin, serotransferrin, and vitamin D binding protein. 2D-DIGE identified 17 proteins that were significantly differentially present in PTL and SPTB. Immunoblotting with specific antibodies confirmed the differences and trends of selected markers. Further characterization and quantification of these markers in a larger cohort of subjects may provide the basis for new tests for the early, noninvasive positive prediction of SPTB.

Comprehensive proteomic analysis of the human amniotic fluid proteome: gestational age-dependent changes.

Amniotic fluid (AF) is a significant contributor to fetal health and constitutes a potential rich source of biomarkers for diagnosis of maternal and fetal disorders. In this study, we performed a comprehensive survey of the proteins expressed in AF, combining gel and liquid-based fractionation approaches coupled with LC-MS/MS analysis. Two-dimensional Liquid Chromatography (2D-LC) analysis identified 118 nonredundant proteins with high confidence. One- and two-dimensional gel electrophoresis and in-gel digestion identified 101 proteins. Combining both sets resulted in 219 proteins, of which 96 are unique to AF; 70, 18, and 35 proteins are present in serum, cervico-vaginal fluid, and all three fluids, respectively. Fluorescence two-dimensional differential in-gel electrophoresis (2D-DIGE) comparison of first-, second-, and third-trimester AF samples revealed that maximal differences in the relative abundance of AF proteins occur between the first and second trimesters. A systematic analysis of proteins present both in AF and maternal serum could lead to the development of new noninvasive diagnostic procedures to monitor fetal status.

Proteomic analysis of cervical-vaginal fluid: identification of novel biomarkers for detection of intra-amniotic infection.

Intra-amniotic infection (IAI) is associated with preterm birth and perinatal mortality. To identify potential biomarkers, we performed a comprehensive survey of the cervical-vaginal fluid (CVF) proteome from a primate IAI model utilizing multidimensional protein identification technology (LC/LC-MS/MS) and MALDI-TOF-MS analyses. Analyses of CVF proteome identified 205 unique proteins and differential expression of 27 proteins in controls and IAI samples. Protein expression signatures and immunodetection of specific biomarkers identified can be employed for noninvasive detection of IAI.

Standardizing global gene expression analysis between laboratories and across platforms.

To facilitate collaborative research efforts between multi-investigator teams using DNA microarrays, we identified sources of error and data variability between laboratories and across microarray platforms, and methods to accommodate this variability. RNA expression data were generated in seven laboratories, which compared two standard RNA samples using 12 microarray platforms. At least two standard microarray types (one spotted, one commercial) were used by all laboratories. Reproducibility for most platforms within any laboratory was typically good, but reproducibility between platforms and across laboratories was generally poor. Reproducibility between laboratories increased markedly when standardized protocols were implemented for RNA labeling, hybridization, microarray processing, data acquisition and data normalization. Reproducibility was highest when analysis was based on biological themes defined by enriched Gene Ontology (GO) categories. These findings indicate that microarray results can be comparable across multiple laboratories, especially when a common platform and set of procedures are used.

GABAergic system gene expression predicts clinical outcome in patients with neuroblastoma.

PURPOSE: Neuroblastoma (NB) is a common childhood malignancy characterized by heterogeneous clinical behavior. The purpose of this study was to identify potential NB biomarkers that may improve outcome prediction. PATIENTS AND METHODS: The suppression subtractive hybridization (SSH) technique was used to identify the genes differentially expressed between NB and control tissue. RNA isolated from 235 primary NB tumor samples obtained from the Children's Cancer Group was evaluated for expression of the candidate markers using quantitative reverse transcriptase polymerase chain reaction (Taqman assays). The association between the mRNA expression levels in the identified candidate genes and clinical outcome was evaluated. RESULTS: SSH analysis identified differential expression of members of the GABAergic gene family in NB. Lower levels of gamma-aminobutyric acid (GABA) receptor-associated protein (GABARAP) gene expression predict decreased survival among all patients. GABA(A) delta receptor subunit gene expression was predictive of a poor outcome among Evans stage IV-S patients. An index of five coexpressed GABA(A) receptor subunits was identified (GABA(A) profile [GAP score]). Patients with a higher GAP score (> -1) had a survival advantage. Multivariate analysis showed that GABARAP and GABA(A) alpha2 receptor subunit gene expression levels and GAP score remained predictors of clinical outcome after accounting for current prognostic indicators. CONCLUSION: Dysregulation of the GABAergic system may constitute a fundamental event in the development of NB, and assessment of GABAergic system gene expression could provide improved patient stratification and potential new therapies.