Isobaric labeling and tandem mass spectrometry: A novel approach for profiling and quantifying proteins differentially expressed in amniotic fluid in preterm labor with and without intra-amniotic infection/inflammation (original) (raw)

2010, Journal of Maternal-Fetal and Neonatal Medicine

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 TM ) 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 ¼ 26); (2) patients who delivered preterm without IAI (n ¼ 25); and (3) patients with IAI (n ¼ 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 MS 2 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 b-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 The iFirst version of this article published online ahead of print (DOI10.3109/14767050903067386) on 6 August 2009 and contained several errors in text citations and reference numbers. The corrected version is currently displayed online. J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by 183.217.172.138 on 05/20/14 For personal use only.