Identification of novel peptides in intra-amniotic infection by proteomics

Abstract

An abstract of a study in the identification of peptides that could be used for sensitive and specific diagnostic tests for intra-amniotic infection (IAI) in rhesus monkeys with experimental IAI and in humans is presented. Here, Gravett et al conclude that the proteome profile described with experimental primate IAI was 100% sensitive and 91% specific in detecting subclinical IAI among a human cohort in preterm labor.

Objective: Intra-amniotic infection (IAI) is a common occurrence in pregnancy and is associated with preterm birth and other adverse neonatal sequelae. However, the early diagnosis of IAI is difficult and depends upon insensitive or non-specific tests. We followed an amniotic fluid (AF) proteomics-based analysis to identify peptides that could be used for sensitive and specific diagnostic tests for IAI in rhesus monkeys with experimental IAI and in humans.

Study design: Surface-enhanced laser desorption ionization (SELDI-TOF, Ciphergen Biosystems), one- and twodimensional gel electrophoresis, and tandem mass spectrometry (MS/MS) were used to characterize AF protein expression in 7 chronically instrumented pregnant rhesus monkeys before and after experimental IAI with group B streptococci1. Automated analysis of MS/MS spectra was performed with SEQUEST software (ThermoFinnigan) and de novo sequencing of all spectra with Luteflsk 1900 v 1.2.5 software. AF protein profiles identified in experimental IAI were then tested in a cohort of 33 women with preterm delivery associated with subclinical IAI (n = II), preterm delivery without IAI (n = II), and preterm contractions with subsequent term delivery (n = II).

Results: Detailed SELDI-TOF spectra revealed differential peptide expression between infected and non-infected primate AF in the 3-5 kD and in the 10-12 kD regions. An 11 kD peak was seen in all animals after infection and in no animal before infection. This 11 kD peak was observed 12 h after infection and preceded increases in uterine contractions or maternal signs and symptoms of IAI. In the human cohort, the 11 kD peak was also identified in II/II patients with subclinical IAI, in 2/11 with preterm delivery without IAI, and in 0/1 I with preterm labor and term delivery without infection (p<0.01). Protein identification within the I I kD peak was obtained by in-gel trypsin digestion and MS/MS analysis. Proteins identified included azuricidin, calgranulin-B, S100 calcium-binding protein, and a unique fragment of insulin growth factor-binding peptide-1 (IGF-BPI). Western blot analysis confirmed the differential expression of these peptides in the setting of infection.

Conclusion: This is the first observation of differentially expressed proteins within AF in the setting of IAI using proteomics-based analysis. The proteome profile described with experimental primate IAI was 100% sensitive and 91% specific in detecting subclinical IAI among a human cohort in preterm labor. We identified differentially expressed proteins that have not been previously associated with IAI but which have immunoregulatory properties. These proteins can potentially be used in the early detection of IAI.

Supported by NIHAI42490, HD06159

Footnote

1. Gravett M. Am J Obstet Gynecol 1994;17:1660-7