Abstract
BackgroundProgress in the fields of protein separation and identification technologies has accelerated research into biofluids proteomics for protein biomarker discovery. Urine has become an ideal and rich source of biomarkers in clinical proteomics. Here we performed a proteomic analysis of urine samples from pregnant and non-pregnant patients using gel electrophoresis and high-resolution mass spectrometry. Furthermore, we also apply a non-prefractionation quantitative phosphoproteomic approach using mTRAQ labeling to evaluate the expression of specific phosphoproteins during pregnancy comparison with non-pregnancy.ResultsIn total, 2579 proteins (10429 unique peptides) were identified, including 1408 from the urine of pregnant volunteers and 1985 from the urine of non-pregnant volunteers. One thousand and twenty-three proteins were not reported in previous studies at the proteome level and were unique to our study. Furthermore, we obtained 237 phosphopeptides, representing 105 phosphoproteins. Among these phosphoproteins, 16 of them were found to be significantly differentially expressed, of which 14 were up-regulated and two were down-regulated in urine samples from women just before vaginal delivery.ConclusionTaken together, these results offer a comprehensive urinary proteomic profile of healthy women during before and after vaginal delivery and novel information on the phosphoproteins that are differentially regulated during the maintenance of normal pregnancy. Our results may provide a better understanding of the mechanisms of pregnancy maintenance, potentially leading to the development of biomarker-based sensitive assays for understanding pregnancy.
Highlights
Progress in the fields of protein separation and identification technologies has accelerated research into biofluids proteomics for protein biomarker discovery
Quantitative differences in protein expression have been detected during pregnancy, which have been useful for the detection of biomarkers for pregnancy-related conditions, such as the identification of fetuses with Down syndrome and preeclampsia, among others [10]
We used a 3 kDa membrane ultrafiltration unit to minimize protein loss and, more importantly, remove low molecular weight polypeptides, which are abundant in human urine samples [17]
Summary
Progress in the fields of protein separation and identification technologies has accelerated research into biofluids proteomics for protein biomarker discovery. Urine can provide proteins for proteomic profile of specific physiological conditions, such as pregnancy, that may be. As it is well known, pregnancy affects protein expression in maternal serum and urine. The resulting peptides were sufficiently resolved by a 5 h gradient using an LTQ Orbitrap analyzer for protein identification and quantification. This method demonstrates that the combination of long-gradient ultra-high pressure liquid chromatography (UHPLC) with high resolution MS at increased sequencing speeds enables extensive proteomic analysis in single runs [15,16]
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