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Background The renin‐angiotensin system plays a crucial role in human physiology, and its main hormone, angiotensin, activates 2 G‐protein–coupled receptors, the angiotensin type‐1 and type‐2 receptors, in almost every organ. However, controversy exists about the location, distribution, and expression levels of these receptors. Concerns have been raised over the low sensitivity, low specificity, and large variability between lots of commercially available antibodies for angiotensin type‐1 and type‐2 receptors, which makes it difficult to reconciliate results of different studies. Here, we describe the first non–antibody‐based sensitive and specific targeted quantitative mass spectrometry assay for angiotensin receptors. Methods and Results Using a technique that allows targeted analysis of multiple peptides across multiple samples in a single mass spectrometry analysis, known as TOMAHAQ (triggered by offset, multiplexed, accurate mass, high resolution, and absolute quantification), we have identified and validated specific human tryptic peptides that permit identification and quantification of angiotensin type‐1 and type‐2 receptors in biological samples. Several peptide sequences are conserved in rodents, making these mass spectrometry assays amenable to both preclinical and clinical studies. We have used this method to quantify angiotensin type‐1 and type‐2 receptors in postmortem frontal cortex samples of older adults (n=28) with Alzheimer dementia. We correlated levels of angiotensin receptors to biomarkers classically linked to renin‐angiotensin system activation, including oxidative stress, inflammation, amyloid‐β load, and paired helical filament‐tau tangle burden. Conclusions These robust high‐throughput assays will not only catalyze novel mechanistic studies in the angiotensin research field but may also help to identify patients with an unbalanced angiotensin receptor distribution who would benefit from angiotensin receptor blocker treatment.

Summary: Methionine adenosyltransferase 1a (MAT1A) is responsible for hepatic S-adenosyl-L-methionine (SAMe) biosynthesis. Mat1a−/− mice have hepatic SAMe depletion, develop nonalcoholic steatohepatitis (NASH) which is reversed with SAMe administration. We examined temporal alterations in the proteome/phosphoproteome in pre-disease and NASH Mat1a−/− mice, effects of SAMe administration, and compared to human nonalcoholic fatty liver disease (NAFLD). Mitochondrial and peroxisomal lipid metabolism proteins were altered in pre-disease mice and persisted in NASH Mat1a−/− mice, which exhibited more progressive alterations in cytoplasmic ribosomes, ER, and nuclear proteins. A common mechanism found in both pre-disease and NASH livers was a hyperphosphorylation signature consistent with casein kinase 2α (CK2α) and AKT1 activation, which was normalized by SAMe administration. This was mimicked in human NAFLD with a metabolomic signature (M-subtype) resembling Mat1a−/− mice. In conclusion, we have identified a common proteome/phosphoproteome signature between Mat1a−/− mice and human NAFLD M-subtype that may have pathophysiological and therapeutic implications.

The identification of extracellular vesicles (EVs) as intercellular conveyors of biological information has recently emerged as a novel paradigm in signaling, leading to the exploitation of EVs and their contents as biomarkers of various diseases. However, whether there are diurnal variations in the size, number, and tissue of origin of blood EVs is currently not known, and could have significant implications when using EVs as biomarkers for disease progression. Currently available technologies for the measurement of EV size and number are either time consuming, require specialized equipment, or lack sufficient accuracy across a range of EV sizes. Flow cytometry represents an attractive alternative to these methods; however, traditional flow cytometers are only capable of measuring particles down to 500 nm, which is significantly larger than the average and median sizes of plasma EVs. Utilizing a Beckman Coulter MoFlo XDP flow cytometer with NanoView module, we employed nanoscale flow cytometry (termed nanoFCM) to examine the relative number and scatter distribution of plasma EVs at three different time points during the day in 6 healthy adults. Analysis of liposomes and plasma EVs proved that nanoFCM is capable of detecting biologically-relevant vesicles down to 100 nm in size. With this high resolution configuration, we observed variations in the relative size (FSC/SSC distributions) and concentration (proportions) of EVs in healthy adult plasma across the course of a day, suggesting that there are diurnal variations in the number and size distribution of circulating EV populations. The use of nanoFCM provides a valuable tool for the study of EVs in both health and disease; however, additional refinement of nanoscale flow cytometric methods is needed for use of these instruments for quantitative particle counting and sizing. Furthermore, larger scale studies are necessary to more clearly define the diurnal variations in circulating EVs, and thus further inform their use as biomarkers for disease.