Human coelomic fluid investigation: A MS-based analytical approach to prenatal screening

Abstract

Coelomic fluid (CF) is the earliest dynamic and complex fluid of the gestational sac. CF contains maternal cells and proteins produced by embryonic cells, tissues and excretions. The biochemical composition of CF is modified throughout the first trimester of pregnancy and its protein profile reflects both physiological/pathological changes affecting the embryo and mother. Identification of variations in the balance of proteins might indicate particular types of pathologies, or ascertain specific genetic disorders. A platform utilizing protein enrichment procedures coupled with shotgun identification and iTRAQ differentiation provided the identification and quantitation of 88 unique embryonic proteins. It is relevant to note that chromosome X protein CXorf23 was found suggesting the embryo sex. Foetal sex was determined by Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR) on coelomic cells, foetal tissues and maternal white blood cells, with a 100% concordance rate between iTRAQ-MS/MS and QF-PCR data. The functional associations among the identified proteins were investigated using STRING database. Open Targets Platform showed as significant the following therapeutic areas: nervous, respiratory, eye and head system disease.

Figure 1

Linear MALDI MS of CF sample.

Scheme 1

Sample preparation protocols used in this study. Protocol I: chemical fractionation; Protocol II: HTP purification; Protocols III: immunodepletion. All fractions appearing in blue were monitored by SDS-PAGE and/or linear MALDI MS.

Figure 2

Linear MALDI spectra of CF from Protocol III_a MARS: (A) after the depletion procedure, and (B) after the deglycosylation step by PNGase F.

Figure 3

Coelomic fluid of sample A. Evaluation of CF for sex determination by quantitative fluorescent PCR. Electropherograms of amplification product from CF without maternal contamination using small tandem repeat (STR) markers specific for chromosome X. A part of the total electropherograms is displayed in Panel A–C (A) coelomic cells pattern; (B) foetal tissue pattern; (C) maternal pattern. AMXY: is present with a peak of 105 bp; DXS1187: STR located in the X chromosome. Coelomic cell DNA and foetal DNA showed two X peaks, one of maternal origin and one of paternal origin. HPRT: STR located in the X chromosome. Coelomic cell DNA and foetal DNA showed two X peaks, one of maternal origin and one of paternal origin.

Figure 4

Coelomic fluid of sample B. Evaluation of CF for sex determination by quantitative fluorescent PCR. Electropherograms of amplification product from CF without maternal contamination using small tandem repeat (STR) markers specific for chromosome Y. A part of the total electropherograms are displayed in Panel A–C (A) coelomic cell pattern; (B) foetal tissue pattern; (C) maternal pattern. AMXY: are visible two different peaks, one peak of 105 bp specific for X chromosome, and one of 110 bp specific for Y chromosome. DXS1187: STR located in the X chromosome. Coelomic cell DNA and foetal DNA showed only one peak of maternal origin. HPRT: STR located in the X chromosome. Coelomic cell DNA and foetal DNA showed only one peak of maternal origin. SRY: STR located in the Y chromosome, absent in the profile of the mother and present as one peak in the profiles of coelomic cell DNA and foetal DNA.

Figure 5

Interaction network analysis of proteins identified by iTRAQ-MARS MS/MS approach (STRING database).

Figure 6

Open Target Platform output. (A) Summary page for 49 targets. (B) Platform workflow for nervous system diseases.