Optimized Targeted Proteomics Methodology for Normalizing Tissue Protein Abundance Data in Analysis of Placental Markers

Abstract

Objective Placental tissue analysis provides important information about the in-utero environment that could have an impact on fetal development. Due to the heterogeneity and variability of perfusion in placental tissue, the technical variability during sample collection can confound placental proteomics data analysis. The aim of this study was to develop a targeted proteomics method for selecting housekeeping placental proteins for normalizing placental data. Methods A list of placental housekeeping proteins was selected based on previously reported placental house-keeping genes (mRNA) (1). The surrogate peptides of the housekeeping proteins were selected for targeted analysis based on an optimized strategy (2). Placental tissue (n=5) was collected from women enrolled in the CANDLE (Conditions Affecting Neurocognitive Development and Learning in Early childhood) study. The samples were fractionated into cytosolic and membrane fractions, and the fractions were enriched, reduced, alkylated, and digested based on previously reported methods (2). Targeted proteomics data were acquired on Waters XevoTQ-XS with IonKey and a microflow liquid chromatography in multiple reaction monitoring mode. The results were analyzed using Skyline software. The coefficient of variation (%CV) of the proteins was calculated to determine the technical or inter-individual variability. Results The following surrogate peptides of the housekeeping proteins were reproducibly quantified: VIGGDDLSTLTGK (HPRT); LGANSLLDLVVFGR (SDHA); LGFQVIFTDFK (TBPL1); DSTLIMQLLR (1433Z); DLLHVLAFSK (LEP); GPQLAAQNLGISLANLLLSK (HEM3); VIHDNFGIVEGLMTTVHAITATQK (G3P); SVALAVLALLSLSGLEAIQR (B2MG); NLVQEWLAK (PPB1); VFDEFKPLVEEPQNLIK (ALBUMIN); and IVEIPFNSTNK (AT1A1), with the fragments and peptides correlation (R2) equal to 0.8-0.99 and 0.80-0.96, respectively. HPRT, SDHA, 1433Z, HEM3, G3P, and B2MG were enriched in the cytosolic fraction, whereas only PPB1 and AT1A1 were enriched in the membrane fraction. TBPL1 and LEP were detected in both fractions. The following proteins showed %CV below 20%: HPRT, SDHA, TBPL1, HEM3, and AT1A1. Positive correlation was observed between 1433Z and G3P, and HPRT and B2MG with an R2 equal to 0.98 and 0.86, respectively. HPRT and SDHA were negatively correlated with an R2 equal to 0.95. Discussion We confirmed that housekeeping proteins could be quantified with good correlation and low coefficients of variation. The placenta is an important organ and its research applications in birth cohort-based studies requires consistent data normalization techniques. Proteomics analysis of placental tissue is paramount to understand the placental phenotype since the proteome is closely associated with function. Creating a cocktail method of housekeeping protein markers that can be used to normalize the protein concentration is vital to comparing samples and data within and across laboratories.