Dataset Information

An Integrated Quantitative Proteomics Workflow for Biomarker Discovery and Validation in Plasma

ABSTRACT: Blood plasma is one of the most widely used samples for biomarker discovery research as well as clinical investigations for diagnostic and therapeutic purposes. However, the plasma proteome is extremely complex due to its wide dynamic range of protein concentrations and the presence of high-abundance proteins. Here we are describing an optimized integrated quantitative proteomics pipeline combining the label-free and multiplexed-labeling-based (Isobaric tags for relative and absolute quantitation) proteome profiling methods for biomarker discovery, followed by the targeted approaches for validation of the identified potential marker proteins. In this workflow, the targeted quantitation of proteins is carried out by multiple-reaction monitoring (MRM) and parallel-reaction monitoring (PRM) mass spectrometry. Thus, our approach enables both unbiased screenings of biomarkers and their subsequent selective validation in human plasma. The overall procedure takes only 1–2 days to complete including the time for data acquisition (excluding database searching). This protocol is quick, flexible and eliminates the need for a separate immunoassay-based validation workflow in blood biomarker investigations.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

SUBMITTER: Vipin Kumar

LAB HEAD: Prof. Sanjeeva Srivastava

PROVIDER: PXD017834 | Pride | 2020-09-07

REPOSITORIES: Pride

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Project description:Low fertility remains a leading cause of poor productivity in dairy cattle. In this context, there is significant interest in developing novel tools for accurate early diagnosis of pregnancy. MicroRNAs (miRNAs) are short RNA molecules which are critically involved in regulating gene expression during both health and disease. MiRNAs have been shown to regulate ovarian function, uterine receptivity, embryonic development and placental function. Circulating miRNAs can provide useful biomarkers of tissue function and disease; importantly, differential miRNA profiles have been linked to pregnancy and preeclampsia in humans. This study sought to establish the potential of circulating miRNAs as biomarkers of early pregnancy in cattle. We applied Illumina small-RNA sequencing to profile miRNAs in plasma samples collected from eight non-pregnant heifers on Days 0, 8 and 16 of the oestrous cycle and 11 heifers on Days 16 and 24 of pregnancy. We sequenced a total of 46 samples and generated 9.2 million miRNA reads per sample. There were no differences in miRNA read abundance between any of the pregnant and non-pregnant time-points (FDRâ>â0.1). As a complementary approach, we analysed sample pools (3-4 samples/pool) corresponding to Days 0, 8 and 16 of the oestrous cycle and Day 24 of pregnancy (nâ=â3 pools/group) using Qiagen PCR arrays. A total of 16 miRNAs were differentially expressed (FDRâ<â0.1) in plasma between pregnant and non-pregnant animals. RT-qPCR validation using the same plasma samples confirmed that miR-26a was differentially upregulated on Day 16 pregnant relative to non-pregnant heifers (1.7-fold; Pâ=â0.043), whereas miR-1249 tended to be upregulated in Day 16 pregnant heifers (1.6-fold; Pâ=â0.081). Further validation in an independent group of heifers confirmed an increase in plasma miR-26a levels during early pregnancy, which was significant only on Day 24 (2.0-fold; Pâ=â0.027). Through genome-wide analyses we have successfully profiled plasma miRNA populations associated with early pregnancy in cattle. We have identified miR-26a as a potential circulating biomarker of early pregnancy. Sequencing of three sequencial samples from each of eight cycling animals (Days 0, 8 and 16, total 24 samples) and two samples from each of 11 pregnant animals (Days 16 and 24, total 22 samples). Main comparisons were between non-pregnant and pregnant groups

Dataset Information

An Integrated Quantitative Proteomics Workflow for Biomarker Discovery and Validation in Plasma

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