Chorioamnionitis disrupts erythropoietin and melatonin homeostasis through the placental-fetal-brain axis during critical developmental periods

Introduction: Novel therapeutics are emerging to mitigate damage from perinatal brain injury (PBI). Few newborns with PBI suffer from a singular etiology. Most experience cumulative insults from prenatal inflammation, genetic and epigenetic vulnerability, toxins (opioids, other drug exposures, environmental exposure), hypoxia-ischemia, and postnatal stressors such as sepsis and seizures. Accordingly, tailoring of emerging therapeutic regimens with endogenous repair or neuro-immunomodulatory agents for individuals requires a more precise understanding of ligand, receptor-, and non-receptor-mediated regulation of essential developmental hormones. Given the recent clinical focus on neurorepair for PBI, we hypothesized that there would be injury-induced changes in erythropoietin (EPO), erythropoietin receptor (EPOR), melatonin receptor (MLTR), NAD-dependent deacetylase sirtuin-1 (SIRT1) signaling, and hypoxia inducible factors (HIF1α, HIF2α). Specifically, we predicted that EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α alterations after chorioamnionitis (CHORIO) would reflect relative changes observed in human preterm infants. Similarly, we expected unique developmental regulation after injury that would reveal potential clues to mechanisms and timing of inflammatory and oxidative injury after CHORIO that could inform future therapeutic development to treat PBI.Methods: To induce CHORIO, a laparotomy was performed on embryonic day 18 (E18) in rats with transient uterine artery occlusion plus intra-amniotic injection of lipopolysaccharide (LPS). Placentae and fetal brains were collected at 24 h. Brains were also collected on postnatal day 2 (P2), P7, and P21. EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α levels were quantified using a clinical electrochemiluminescent biomarker platform, qPCR, and/or RNAscope. MLT levels were quantified with liquid chromatography mass spectrometry.Results: Examination of EPO, EPOR, and MLTR1 at 24 h showed that while placental levels of EPO and MLTR1 mRNA were decreased acutely after CHORIO, cerebral levels of EPO, EPOR and MLTR1 mRNA were increased compared to control. Notably, CHORIO brains at P2 were SIRT1 mRNA deficient with increased HIF1α and HIF2α despite normalized levels of EPO, EPOR and MLTR1, and in the presence of elevated serum EPO levels. Uniquely, brain levels of EPO, EPOR and MLTR1 shifted at P7 and P21, with prominent CHORIO-induced changes in mRNA expression. Reductions at P21 were concomitant with increased serum EPO levels in CHORIO rats compared to controls and variable MLT levels.Discussion: These data reveal that commensurate with robust inflammation through the maternal placental-fetal axis, CHORIO impacts EPO, MLT, SIRT1, and HIF signal transduction defined by dynamic changes in EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α mRNA, and EPO protein. Notably, ligand-receptor mismatch, tissue compartment differential regulation, and non-receptor-mediated signaling highlight the importance, complexity and nuance of neural and immune cell development and provide essential clues to mechanisms of injury in PBI. As the placenta, immune cells, and neural cells share many common, developmentally regulated signal transduction pathways, further studies are needed to clarify the perinatal dynamics of EPO and MLT signaling and to capitalize on therapies that target endogenous neurorepair mechanisms

Standard Flow Multiplexed Proteomics (SFloMPro)—An Accessible Alternative to NanoFlow Based Shotgun Proteomics

Multiplexed proteomics using isobaric tagging allows for simultaneously comparing the proteomes of multiple samples. In this technique, digested peptides from each sample are labeled with a chemical tag prior to pooling sample for LC-MS/MS with nanoflow chromatography (NanoLC). The isobaric nature of the tag prevents deconvolution of samples until fragmentation liberates the isotopically labeled reporter ions. To ensure efficient peptide labeling, large concentrations of labeling reagents are included in the reagent kits to allow scientists to use high ratios of chemical label per peptide. The increasing speed and sensitivity of mass spectrometers has reduced the peptide concentration required for analysis, leading to most of the label or labeled sample to be discarded. In conjunction, improvements in the speed of sample loading, reliable pump pressure, and stable gradient construction of analytical flow HPLCs has continued to improve the sample delivery process to the mass spectrometer. In this study we describe a method for performing multiplexed proteomics without the use of NanoLC by using offline fractionation of labeled peptides followed by rapid “standard flow” HPLC gradient LC-MS/MS. Standard Flow Multiplexed Proteomics (SFloMPro) enables high coverage quantitative proteomics of up to 16 mammalian samples in about 24 h. In this study, we compare NanoLC and SFloMPro analysis of fractionated samples. Our results demonstrate that comparable data is obtained by injecting 20 µg of labeled peptides per fraction with SFloMPro, compared to 1 µg per fraction with NanoLC. We conclude that, for experiments where protein concentration is not strictly limited, SFloMPro is a competitive approach to traditional NanoLC workflows with improved up-time, reliability and at a lower relative cost per sample

The Open Pesticide Transition Library (oPestTL)

The identification of pesticide residues is a critical application in environmental and food safety analysis. The most common methods for pesticide residue detection and quantification are performed with liquid chromatography tandem mass spectrometry (LCMS) using instruments with varying architectures and characteristics. Pesticides have diverse structural and elemental compositions and often require laborious instrument-specific user optimization to identify ideal adducts and fragment ions. The Open Pesticide Transition Library (oPestTL) is an effort to compile the most comprehensive list of pesticide ion targets and fragments to facilitate the rapid development of screening methods for any LCMS hardware configuration. While a work that must inevitably evolve, the oPestTL library release detailed here contains over 4,000 individual transitions spread across seven separate LCMS platforms making it the largest openly available collection of pesticide methods ever assembled

Better than flipping a coin? Organ specific plasma proteins are not confidently identified by gene expression data

A recent study by Oh et al., utilized plasma proteomic samples to produce an impressive model predictive of human aging at an organ specific level. While both the depth and size of this study are nearly unprecedented, proteins were grouped by the organ they originated from based on gene expression data. This is surprising because it is well accepted that the correlation between protein abundance and transcript abundance is generally poor. It is more surprising that the authors did not leverage protein specific databases such as the Draft Maps of the Human Proteome published in Nature in 2014. Spot checking proteins these “organ specific” proteins against these resources suggested there was generally low levels of specificity. For a thorough analysis I compared all proteins identified as organ specific from a high depth proteomic atlas of 29 healthy human tissues. Of 460 proteins which could be matched to organ specific data between the two studies only 274 (59.6%) met the author’s threshold for organ specificity when evaluating protein level data. When considering human organs as a connected network with equivalent protein level contributions to plasma, only 209 (45.4%) of the organ specific proteins had a greater summed abundance than the other organs. While there may be validity in the use of plasma proteomics to assess human aging, organ specific patterns should be based on protein level data, rather than gene expression for reliable metric

LCMSMethods.org – A curated repository to enhance the sharing and dissemination of mass spectrometry methods

Mass spectrometry (MS) is a rapidly growing field that impacts nearly every branch of science today. Improvements in instrument hardware, separation techniques and data processing have allowed the application of the fundamental principles of ion movement in vacuum chambers to be applied to solve a variety of problems. Many researchers looking to add MS technology to their laboratory are challenged by the fact that most instruments do not arrive with optimized instrument experiments for their application. I present herein the current iteration of LCMS-Methods.org, a project that has gradually evolved since it was first launched in 2014. This brief review describes the motivation, current state, and future goals of this growing resource for the mass spectrometry community

Identifying post-translational modifications with single cell proteomics

With the rapid expansion of capabilities in the analysis of proteins in single cells, we now can now identify multiple classes of protein post-translational modifications on some of these proteins. Each new technology that has increased our protein coverage has likewise increased our ability to identify and quantify modified peptides. In this chapter I will discuss our current capabilities and challenges specific to this emerging field of study and inevitable service of demand