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Objective. Examination of the amniotic fluid (AF) proteome has been previously attempted to identify useful biomarkers in predicting the outcome of preterm labor (PTL). Isobaric Tag for Relative and Absolute Quantitation (iTRAQ™) labeling allows direct ratiometric comparison of relative abundance of identified protein species among multiplexed samples. The purpose of this study was to apply, for the first time, the combination of iTRAQ and tandem mass spectrometry to identify proteins differentially regulated in AF samples of women with spontaneous PTL and intact membranes with and without intra-amniotic infection/inflammation (IAI). Methods. A cross-sectional study was designed and included AF samples from patients with spontaneous PTL and intact membranes in the following groups: (1) patients without IAI who delivered at term ( n = 26); (2) patients who delivered preterm without IAI ( n = 25); and (3) patients with IAI ( n = 24). Proteomic profiling of AF samples was performed using a workflow involving tryptic digestion, iTRAQ labeling and multiplexing, strong cation exchange fractionation, and liquid chromatography tandem mass spectrometry. Twenty-five separate 4-plex samples were prepared and analyzed. Results. Collectively, 123,011 MS2 spectra were analyzed, and over 25,000 peptides were analyzed by database search (X!Tandem and Mascot), resulting in the identification of 309 unique high-confidence proteins. Analysis of differentially present iTRAQ reporter peaks revealed many proteins that have been previously reported to be associated with preterm delivery with IAI. Importantly, many novel proteins were found to be up-regulated in the AF of patients with PTL and IAI including leukocyte elastase precursor, Thymosin-like 3, and 14-3-3 protein isoforms. Moreover, we observed differential expression of proteins in AF of patients who delivered preterm in the absence of IAI in comparison with those with PTL who delivered at term including Mimecan precursor, latent-transforming growth factor β-binding protein isoform 1L precursor, and Resistin. These findings have been confirmed for Resistin in an independent cohort of samples using ELISA. Gene ontology enrichment analysis was employed to reveal families of proteins participating in distinct biological processes. We identified enrichment for host defense, anti-apoptosis, metabolism/catabolism and cell and protein mobility, localization and targeting. Conclusions. (1) Proteomics with iTRAQ labeling is a profiling tool capable of revealing differential expression of proteins in AF; (2) We discovered 82 proteins differentially expressed in three clinical subgroups of premature labor, 67 which were heretofore unknown. Of particular importance is the identification of proteins differentially expressed in AF from women who delivered preterm in the absence of IAI. This is the first report of the positive identification of biomarkers in this subgroup of patients. [ABSTRACT FROM AUTHOR]