Amniotic Fluid Proteome Analysis from Down Syndrome Pregnancies for Biomarker Discovery

Abstract

Down syndrome (DS) is an anomaly caused by an extra chromosome 21, and it affects 1 in 750 live births. Phenotypes include cognitive impairment, congenital defects, and increased risk for several diseases such as Alzheimer's disease and leukemia. Current DS-screening tests subject many women to invasive procedures for accurate diagnosis due to insufficient specificity. Since amniotic fluid (AF) surrounds the developing fetus, understanding the changes in AF composition in the presence of DS may provide insights into genotype-phenotype associations, and aid in discovery of novel biomarkers for better screening. On the basis of our previous study, in which we reported an extensive proteome of AF, we performed two-dimensional liquid chromatography followed by MS/MS to analyze triplicates of pooled AF of chromosomally normal and DS-affected pregnancies (10 samples per pool). A total of 542 proteins were identified from the two sets of triplicate analyses by the LTQ-Orbitrap mass spectrometer and data were compared semiquantitatively by spectral counting. Candidate biomarkers were selected based on the spectral count differences between the two conditions after normalization. Comparison between the two groups revealed 60 candidates that showed greater than 2-fold increase or decrease in concentration in the presence of DS. Among these candidates, amyloid precursor protein and tenascin-C were verified by ELISA, and both showed a 2-fold increase, on average, in DS-AF samples compared to controls. All proteins that showed significant differences between the two conditions were bioinformatically analyzed to preliminarily understand their biological implications in DS.

A proteomic analysis of human follicular fluid: comparison between fertilized oocytes and non-fertilized oocytes in the same patient

“…In LC/MS/MS analyses, the spectral counts of proteins significantly vary with respect to the expression level of each protein [7]. Spectral counting was applied in this study for high-throughput quantification of the total number of spectra.…”

“…However, a limitation of this study is that we are unable to attribute fertilization to oocyte quality only, although we Fig. 2 Spectral counts of heparan sulfate proteoglycan perlecan in the follicular fluids containing fertilized oocytes (fFF) and non-fertilized oocytes (nfFF) in each patient (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) tried to minimize the influence of sperm quality using ICSI. An increased number of samples would enable the use of a proteomics analysis to identify distinctive biomarkers.…”

“…Tandem, Protein Prophet and Peptide Prophet [22] to sequence the MS/MS data. The Scaffold data analysis consisted of a quantitative comparison followed by a 95 % peptide identification probability analysis and a 99 % protein identification probability analysis that contained two or more identified peptides [7]. Proteins that contained similar peptides and could not be differentiated based on an MS/MS analysis alone were grouped together to satisfy the principles of parsimony.…”

Unraveling the sperm proteome and post-genomic pathways associated with sperm nuclear DNA fragmentation

“…These pools were then quantified in triplicate using the BCA assay mentioned above. A comprehensive shotgun analysis was then performed using the pooled samples in an experimental design created along the lines published by Cho et al [34] and Pan et al [35], and four technical replicates of each pool were used.…”

Verification of a biomarker discovery approach for detection of Down syndrome in amniotic fluid via multiplex selected reaction monitoring (SRM) assay

“…Therefore, AF has been proteomically studied in depth to explore its potential as a medium for biomarker discovery [6][7][8]. Furthermore, several studies have attempted to compare AF proteins from chromosomally normal (CN) and DS-affected pregnancies semi-quantitatively and quantitatively [9][10][11]. In our previous study, a comparison of spectral counts of proteins from unaffected and affected (Down syndrome) AF proteome revealed 60 candidates.…”