Beyond the model organism-Mechanistic analysis of protein post-translational modifications is ready for all challengers.

A historic challenge for shotgun proteomics has been the requirement for high quality, simple, and nonredundant curated protein sequences in small .fasta text files. Due to the intrinsic informatic challenges and time required to assemble these files, proteomics has struggled to expand beyond the confines of a few model organisms. When considering post-translational modifications that may or may not be present on a specific peptide sequence, these factors inevitably compound. A study on how mangos continue to ripen on the shelf may not be the first thing you'd think of as proof of a scientific discipline shedding historic limitations. However, Bautiste-Valle et al., may be just that. These authors present a quantitative comparison of both peptide and glycopeptide alterations through the complexity of the fruit ripening process and in this we see the present state of a field that no longer needs to wait on genomics to obtain deep mechanistic insights.

Single cell proteomics by mass spectrometry reveals deep epigenetic insight and new targets of a class specific histone deacetylase inhibitor.

Epigenetic programming has been shown to play a role in nearly every human system and disease where anyone has thought to look. However, the levels of heterogeneity at which epigenetic or epiproteomic modifications occur at single cell resolution across a population remains elusive. While recent advances in sequencing technology have allowed between 1 and 3 histone post-translational modifications to be analyzed in each single cell, over twenty separate chemical PTMs are known to exist, allowing thousands of possible combinations. Single cell proteomics by mass spectrometry (SCP) is an emerging technology in which hundreds or thousands of proteins can be directly quantified in typical human cells. As the proteins detected and quantified by SCP are heavily biased toward proteins of highest abundance, chromatin proteins are an attractive target for analysis. To this end, I applied SCP to the analysis of cancer cells treated with mocetinostat, a class specific histone deacetylase inhibitor. I find that 16 PTMs can be confidently identified and localized with high site specificity in single cells. In addition, the high abundance of histone proteins allows higher throughput methods to be utilized for SCP than ever described. While quantitative accuracy suffers when analyzing more than 700 cells per day, 9 histone proteins can be measured in single cells analyzed at even 3,500 cells per day, a throughput 10-fold greater than any previous report. In addition, the unbiased global approach utilized herein identifies a previously uncharacterized response to this drug through the S100-A8/S100-A9 protein complex partners. This response is observed in nearly every cell of the over 1,000 analyzed in this study, regardless of the relative throughput of the method utilized. While limitations exist in the methods described herein, current technologies can easily improve upon the results presented here to allow comprehensive analysis of histone PTMs to be performed in any mass spectrometry lab. All raw and processed data described in this study has been made publicly available through the ProteomeXchange/MASSIVE repository system as MSV000093434.

Cocaine regulates antiretroviral therapy CNS access through pregnane-x receptor-mediated drug transporter and metabolizing enzyme modulation at the blood brain barrier.

BACKGROUND: Appropriate interactions between antiretroviral therapies (ART) and drug transporters and metabolizing enzymes at the blood brain barrier (BBB) are critical to ensure adequate dosing of the brain to achieve HIV suppression. These proteins are modulated by demographic and lifestyle factors, including substance use. While understudied, illicit substances share drug transport and metabolism pathways with ART, increasing the potential for adverse drug:drug interactions. This is particularly important when considering the brain as it is relatively undertreated compared to peripheral organs and is vulnerable to substance use-mediated damage. METHODS: We used an in vitro model of the human BBB to determine the extravasation of three first-line ART drugs, emtricitabine (FTC), tenofovir (TFV), and dolutegravir (DTG), in the presence and absence of cocaine, which served as our illicit substance model. The impact of cocaine on BBB integrity and permeability, drug transporters, metabolizing enzymes, and their master transcriptional regulators were evaluated to determine the mechanisms by which substance use impacted ART central nervous system (CNS) availability. RESULTS: We determined that cocaine had a selective impact on ART extravasation, where it increased FTC's ability to cross the BBB while decreasing TFV. DTG concentrations that passed the BBB were below quantifiable limits. Interestingly, the potent neuroinflammatory modulator, lipopolysaccharide, had no effect on ART transport, suggesting a specificity for cocaine. Unexpectedly, cocaine did not breach the BBB, as permeability to albumin and 4 kDa FITC-dextran, as well as tight junction proteins and adhesion molecules remained unchanged. Rather, cocaine selectively decreased the pregnane-x receptor (PXR), but not constitutive androstane receptor (CAR). Consequently, drug transporter expression and activity decreased in endothelial cells of the BBB, including p-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 4 (MRP4). Further, cytochrome P450 3A4 (CYP3A4) enzymatic activity increased following cocaine treatment that coincided with decreased expression. Finally, cocaine modulated adenylate kinases that are required to facilitate biotransformation of ART prodrugs to their phosphorylated, pharmacologically active counterparts. CONCLUSION: Our findings indicate that additional considerations are needed in CNS HIV treatment strategies for people who use cocaine, as it may limit ART efficacy through regulation of drug transport and metabolizing pathways at the BBB.

Tenofovir Activation Is Diminished in the Brain and Liver of Creatine Kinase Brain-Type Knockout Mice.

Tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor prescribed for the treatment and prevention of human immunodeficiency virus infection and the treatment of chronic hepatitis B virus infection. Here, we demonstrate that creatine kinase brain-type (CKB) can form tenofovir-diphosphate (TFV-DP), the pharmacologically active metabolite, in vitro and identify nine missense mutations (C74S, R96P, S128R, R132H, R172P, R236Q, C283S, R292Q, and H296R) that diminish this activity. Additional characterization of these mutations reveals that five (R96P, R132H, R236Q, C283S, and R292Q) have ATP dephosphorylation catalytic efficiencies less than 20% of those of the wild type (WT), and seven (C74S, R96P, R132H, R172P, R236Q, C283S, and H296P) induce thermal instabilities. To determine the extent CKB contributes to TFV activation in vivo, we generated a CKB knockout mouse strain, Ckbtm1Nnb. Using an in vitro assay, we show that brain lysates of Ckbtm1Nnb male and female mice form 70.5 and 77.4% less TFV-DP than wild-type brain lysates of the same sex, respectively. Additionally, we observe that Ckbtm1Nnb male mice treated with tenofovir disoproxil fumarate for 14 days exhibit a 22.8% reduction in TFV activation in the liver compared to wild-type male mice. Lastly, we utilize mass spectrometry-based proteomics to elucidate the impact of the knockout on the abundance of nucleotide and small molecule kinases in the brain and liver, adding to our understanding of how the loss of CKB may be impacting tenofovir activation in these tissues. Together, our data suggest that disruptions in CKB may lower levels of active drugs in the brain and liver.

Spatial Heterogeneity of Brain Lipids in SIV-Infected Macaques Treated with Antiretroviral Therapy.

Human immunodeficiency virus (HIV) infection continues to promote neurocognitive impairment, mood disorders, and brain atrophy, even in the modern era of viral suppression. Brain lipids are vulnerable to HIV-associated energetic strain and may contribute to HIV-associated neurologic dysfunction due to alterations in lipid breakdown and structural lipid composition. HIV neuropathology is region dependent, yet there has not been comprehensive characterization of the spatial heterogeneity of brain lipids during infection that possibly impacts neurologic function. To address this gap, we evaluated the spatial lipid distribution using matrix laser desorption/ionization imaging mass spectrometry (MALDI-IMS) across four brain regions (parietal cortex, midbrain, thalamus, and temporal cortex), as well as the kidney for a peripheral tissue control, in a simian immunodeficiency virus (SIV)-infected rhesus macaque treated with a course of antiretroviral therapies (ARTs). We assessed lipids indicative of fat breakdown [acylcarnitines (CARs)] and critical structural lipids [phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs)] across fatty acid chain lengths and degrees of unsaturation. CARs with very long-chain, polyunsaturated fatty acids (PUFAs) were more abundant across all brain regions than shorter chain, saturated, or monounsaturated species. We observed distinct brain lipid distribution patterns for the CARs and PCs. However, no clear expression patterns emerged for PEs. Surprisingly, the kidney was nearly devoid of ions corresponding to PUFAs common in brain. PEs and PCs with PUFAs had little intensity and less density than other species, and only one CAR species was observed in kidney at high intensity. Overall, our study demonstrates the stark variation in structural phospholipids and lipid-energetic intermediates present in the virally suppressed SIV-macaque brain. These findings may be useful for identifying regional vulnerabilities to damage due to brain lipid changes in people with HIV.

Metabolomic, Proteomic, and Single-Cell Proteomic Analysis of Cancer Cells Treated with the KRASG12D Inhibitor MRTX1133.

Mutations in KRAS are common drivers of human cancers and are often those with the poorest overall prognosis for patients. A recently developed compound, MRTX1133, has shown promise in inhibiting the activity of KRASG12D mutant proteins, which is one of the main drivers of pancreatic cancer. To better understand the mechanism of action of this compound, I performed both proteomics and metabolomics on four KRASG12D mutant pancreatic cancer cell lines. To obtain increased granularity in the proteomic observations, single-cell proteomics was successfully performed on two of these lines. Following quality filtering, a total of 1498 single cells were analyzed. From these cells, 3140 total proteins were identified with approximately 953 proteins quantified per cell. At 48 h of treatment, two distinct populations of cells can be observed based on the level of effectiveness of the drug in decreasing the total abundance of the KRAS protein in each respective cell, with results that are effectively masked in the bulk cell analysis. All mass spectrometry data and processed results are publicly available at www.massive.ucsd.edu at accessions PXD039597, PXD039601, and PXD039600.

Simple Tool for Rapidly Assessing the Quality of Multiplexed Single Cell Proteomics Data.

Recent advances in the sensitivity and speed of mass spectrometers coupled with improved sample preparation methods have enabled the field of single cell proteomics to proliferate. While heavy development is occurring in the label free space, dramatic improvements in throughput are provided by multiplexing with tandem mass tags. Hundreds or thousands of single cells can be analyzed with this method, yielding large data sets which may contain poor data arising from loss of material during cell sorting or poor digestion, labeling, and lysis. To date, no tools have been described that can assess data quality prior to data processing. We present herein a lightweight python script and accompanying graphic user interface that can rapidly quantify reporter ion peaks within each MS/MS spectrum in a file. With simple summary reports, we can identify single cell samples that fail to pass a set quality threshold, thus reducing analysis time waste. In addition, this tool, Diagnostic Ion Data Analysis Reduction (DIDAR), will create reduced MGF files containing only spectra possessing a user-specified number of single cell reporter ions. By reducing the number of spectra that have excessive zero values, we can speed up sample processing with little loss in data completeness as these spectra are removed in later stages in data processing workflows. DIDAR and the DIDAR GUI are compatible with all modern operating systems and are available at: https://github.com/orsburn/DIDARSCPQC. All files described in this study are available at www.massive.ucsd.edu as accession MSV000088887.

An integrated method for single cell proteomics with simultaneous measurements of intracellular drug concentration implicates new mechanisms for adaptation to KRASG12D inhibitors.

It is well established that a population of single human cells will often respond to the same drug treatment in a heterogeneous manner. In the context of chemotherapeutics, these diverse responses may lead to individual adaptation mechanisms and ultimately multiple distinct methods of resistance. The obvious question from a pharmacology perspective is how intracellular concentrations of active drug varies between individual cells, and what role does that variation play in drug response heterogeneity? To date, no integrated methods for rapidly measuring intracellular drug levels while simultaneously measuring drug responses have been described. This study describes a method for single cell preparation that allows proteins to be extracted and digested from single cells while maintaining conditions for small molecules to be simultaneously measured. The method as described allows up to 40 cells to be analyzed per instrument per day. When applied to a KRASG12D small molecule inhibitor I observe a wide degree of intracellular levels of the drug, and that proteomic responses largely stratify based on the concentration of drug within each single cell. Further work is in progress to develop and standardize this method and - more importantly - to normalize drug measurements against direct measurements of cell volume. However, these preliminary results appear promising for the identification of single cells with unique drug response mechanisms. All data described in this study has been made publicly available through the ProteomeXchange consortium under accession PXD046002.

Tenofovir Activation is Diminished in the Brain and Liver of Creatine Kinase Brain-Type Knockout Mice.

Tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor prescribed for the treatment and prevention of human immunodeficiency virus infection, and the treatment of chronic hepatitis B virus infection. Here, we demonstrate that creatine kinase brain-type (CKB) can form tenofovir-diphosphate (TFV-DP), the pharmacologically active metabolite, in vitro, and identify nine missense mutations (C74S, R96P, S128R, R132H, R172P, R236Q, C283S, R292Q, and H296R) that diminish this activity. Additional characterization of these mutations reveal that five (R96P, R132H, R236Q, C283S, and R292Q) have ATP dephosphorylation catalytic efficiencies less than 20% of wild-type (WT), and seven (C74S, R96P, R132H, R172P, R236Q, C283S, and H296P) induce thermal instabilities. To determine the extent CKB contributes to TFV activation in vivo, we generated a CKB knockout mouse strain, Ckbtm1Nnb. Using an in vitro assay, we show that brain lysates of Ckbtm1Nnb male and female mice form 70.5% and 77.4% less TFV-DP than wild-type brain lysates of the same sex, respectively. Additionally, we observe that Ckbtm1Nnb male mice treated with tenofovir disoproxil fumarate for 14 days exhibit a 22.8% reduction in TFV activation in liver compared to wild-type male mice. Lastly, we utilize mass spectrometry-based proteomics to elucidate the impact of the knockout on the abundance of nucleotide and small molecule kinases in the brain and liver, adding to our understanding of how loss of CKB may be impacting tenofovir activation in these tissues. Together, our data suggest that disruptions in CKB may lower levels of active drug in brain and liver.

SCP Viz - A universal graphical user interface for single protein analysis in single cell proteomics datasets.

Single cell proteomics (SCP) requires the analysis of dozens to thousands of single human cells to draw biological conclusions. However, assessing of the abundance of single proteins in output data presents a considerable challenge, and no simple universal solutions currently exist. To address this, we developed SCP Viz, a statistical package with a graphical user interface that can handle small and large scale SCP output from any instrument or data processing software. In this software, the abundance of individual proteins can be plotted in a variety of ways, using either unadjusted or normalized outputs. These outputs can also be transformed or imputed within the software. SCP Viz offers a variety of plotting options which can help identify significantly altered proteins between groups, both before and after quantitative transformations. Upon the discovery of subpopulations of single cells, users can easily regroup the cells of interest using straightforward text-based filters. When used in this way, SCP Viz allows users to visualize proteomic heterogeneity at the level of individual proteins, cells, or identified subcellular populations. SCP Viz is compatible with output files from MaxQuant, FragPipe, SpectroNaut, and Proteome Discoverer, and should work equally well with other formats. SCP Viz is publicly available at https://github.com/orsburn/SCPViz. For demonstrations, users can download our test data from GitHub and use an online version that accepts user input for analysis at https://orsburnlab.shinyapps.io/SCPViz/.

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.

Cocaine Regulates Antiretroviral Therapy CNS Access Through Pregnane-X Receptor-Mediated Drug Transporter and Metabolizing Enzyme Modulation at the Blood Brain Barrier.

Background: Appropriate interactions between antiretroviral therapies (ART) and drug transporters and metabolizing enzymes at the blood brain barrier (BBB) are critical to ensure adequate dosing of the brain to achieve HIV suppression. These proteins are modulated by demographic and lifestyle factors, including substance use. While understudied, illicit substances share drug transport and metabolism pathways with ART, increasing the potential for adverse drug:drug interactions. This is particularly important when considering the brain as it is relatively undertreated compared to peripheral organs and is vulnerable to substance use-mediated damage. Methods: We used an in vitro model of the human BBB to determine the extravasation of three first-line ART drugs, emtricitabine (FTC), tenofovir (TFV), and dolutegravir (DTG), in the presence and absence of cocaine, which served as our illicit substance model. The impact of cocaine on BBB integrity and permeability, drug transporters, metabolizing enzymes, and their master transcriptional regulators were evaluated to determine the mechanisms by which substance use impacted ART central nervous system (CNS) availability. Results: We determined that cocaine had a selective impact on ART extravasation, where it increased FTC's ability to cross the BBB while decreasing TFV. DTG concentrations that passed the BBB were below quantifiable limits. Interestingly, the potent neuroinflammatory modulator, lipopolysaccharide, had no effect on ART transport, suggesting a specificity for cocaine. Unexpectedly, cocaine did not breach the BBB, as permeability to albumin and tight junction proteins and adhesion molecules remained unchanged. Rather, cocaine selectively decreased the pregnane-x receptor (PXR), but not constitutive androstane receptor (CAR). Consequently, drug transporter expression and activity decreased in endothelial cells of the BBB, including p-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 4 (MRP4). Further, cytochrome P450 3A4 (CYP3A4) enzymatic activity increased following cocaine treatment that coincided with decreased expression. Finally, cocaine modulated adenylate kinases are required to facilitate biotransformation of ART prodrugs to their phosphorylated, pharmacologically active counterparts. Conclusion: Our findings indicate that additional considerations are needed in CNS HIV treatment strategies for people who use cocaine, as it may limit ART efficacy through regulation of drug transport and metabolizing pathways at the BBB.

Acetic acid is a superior acidifier for sub-nanogram and single cell proteomic studies.

A recent study demonstrated a substantial signal increase when employing a 0.5% acetic acid buffer additive instead of the traditional 0.1% formic acid used in shotgun proteomics. In this study I compare these two buffers for a dilution series of tryptic digests down to 20 picograms peptide on column on a TIMSTOF single cell proteome (SCP) system. I observe a comparable relative level of signal increase as previously reported, which translates to improvements in proteome coverage at every peptide load assessed. The relative increase in peptide identifications is more apparent at lower concentrations with a striking 1.8-fold more peptides identified at 20 pg peptide load, resulting in over 2,000 protein groups identified in 30 minutes on this system. These results translate well to isolated single human cancer cells allowing over 1,000 protein groups to be identified in single human cells processed using a simple one step method in standard 96-well plates. All vendor raw and processed data has been made publicly available at www.massive.ucsd.edu and can be accessed as MSV000092563.

Metabolomic, proteomic and single cell proteomic analysis of cancer cells treated with the KRASG12D inhibitor MRTX1133.

Mutations in KRAS are common drivers of human cancers and are often those with the poorest overall prognosis for patients. A recently developed compound, MRTX1133, has shown promise in inhibiting the activity of KRASG12D mutant proteins, one of the main drivers in pancreatic cancer. To better understand the mechanism of action of this compound I performed both proteomics and metabolomics on four KRASG12D mutant pancreatic cancer cell lines. To obtain increased granularity in the proteomic observations, single cell proteomics was successfully performed on two of these lines. Following quality filtering, a total of 1,498 single cells were analyzed. From these cells 3,140 total proteins were identified with approximately 953 proteins quantified per cell. At 48 hours of treatment, two distinct populations of cells can be observed based on the level of effectiveness of the drug in decreasing total abundance of the KRAS protein in each respective cell, results that are effectively masked in the bulk cell analysis. All mass spectrometry data and processed results are publicly available at the www.massive.ucsd.edu at accessions PXD039597, PXD039601 and PXD039600.

The SAGA HAT module is tethered by its SWIRM domain and modulates activity of the SAGA DUB module.

The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is a transcriptional co-activator that both acetylates and deubiquitinates histones. The histone acetyltransferase (HAT) subunit, Gcn5, is part of a subcomplex of SAGA called the HAT module. A minimal HAT module complex containing Gcn5 bound to Ada2 and Ada3 is required for full Gcn5 activity on nucleosomes. Deletion studies have suggested that the Ada2 SWIRM domain plays a role in tethering the HAT module to the remainder of SAGA. While recent cryo-EM studies have resolved the structure of the core of the SAGA complex, the HAT module subunits and molecular details of its interactions with the SAGA core could not be resolved. Here we show that the SWIRM domain is required for incorporation of the HAT module into the yeast SAGA complex, but not the ADA complex, a distinct six-protein acetyltransferase complex that includes the SAGA HAT module proteins. In the isolated Gcn5/Ada2/Ada3 HAT module, deletion of the SWIRM domain modestly increased activity but had negligible effect on nucleosome binding. Loss of the HAT module due to deletion of the SWIRM domain decreases the H2B deubiquitinating activity of SAGA, indicating a role for the HAT module in regulating SAGA DUB module activity. A model of the HAT module created with Alphafold Multimer provides insights into the structural basis for our biochemical data, as well as prior deletion studies.

Biotransformation of Efavirenz and Proteomic Analysis of Cytochrome P450s and UDP-Glucuronosyltransferases in Mouse, Macaque, and Human Brain-Derived In Vitro Systems.

Antiretroviral drugs such as efavirenz (EFV) are essential to combat human immunodeficiency virus (HIV) infection in the brain, but little is known about how these drugs are metabolized locally. In this study, the cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT)-dependent metabolism of EFV was probed in brain microsomes from mice, cynomolgus macaques, and humans as well as primary neural cells from C57BL/6N mice. Utilizing ultra high performance liquid chromatography high-resolution mass spectrometry (uHPLC-HRMS), the formation of 8-hydroxyefavirenz (8-OHEFV) from EFV and the glucuronidation of P450-dependent metabolites 8-OHEFV and 8,14-dihydroxyefavirenz (8,14-diOHEFV) were observed in brain microsomes from all three species. The direct glucuronidation of EFV, however, was only detected in cynomolgus macaque brain microsomes. In primary neural cells treated with EFV, microglia were the only cell type to exhibit metabolism, forming 8-OHEFV only. In cells treated with the P450-dependent metabolites of EFV, glucuronidation was detected only in cortical neurons and astrocytes, revealing that certain aspects of EFV metabolism are cell type specific. Untargeted and targeted proteomics experiments were used to identify the P450s and UGTs present in brain microsomes. Eleven P450s and 11 UGTs were detected in human brain microsomes, whereas seven P450s and 14 UGTs were identified in mouse brain microsomes and 15 P450s and four UGTs, respectively, were observed in macaque brain microsomes. This was the first time many of these enzymes have been noted in brain microsomes at the protein level. This study indicates the potential for brain metabolism to contribute to pharmacological and toxicological outcomes of EFV in the brain. SIGNIFICANCE STATEMENT: Metabolism in the brain is understudied, and the persistence of human immunodeficiency virus (HIV) infection in the brain warrants the evaluation of how antiretroviral drugs such as efavirenz are metabolized in the brain. Using brain microsomes, the metabolism of efavirenz by both cytochrome P450s (P450s) and UDP-glucuronosyltransferases (UGTs) is established. Additionally, proteomics of brain microsomes characterizes P450s and UGTs in the brain, many of which have not yet been noted in the literature at the protein level.

Time-of-Flight Fragmentation Spectra Generated by the Proteomic Analysis of Single Human Cells Do Not Exhibit Atypical Fragmentation Patterns.

Recent work detailed the unique characteristics of fragmentation spectra derived from peptides from single human cells. This valuable report utilized an ultrahigh-field Orbitrap and directly compared the spectra obtained from high-concentration bulk cell HeLa lysates to those obtained from nanogram dilutions of the same and from nanowell-processed single HeLa cells. The analysis demonstrated marked differences between the fragmentation spectra generated at high and single-cell loads, most strikingly, the loss of high-mass y-series fragment ions. As significant differences exist in the physics of Orbitrap and time-of-flight mass analyzers, a comparison appeared warranted. A similar analysis was performed using isolated single pancreatic cancer cells compared to pools consisting of 100 cells. While a reanalysis of the prior Orbitrap data supports the author's original findings, the same trends are not observed in time-of-flight mass spectra of peptides from single human cells. The results are particularly striking when directly comparing the matched intensity fragment values between bulk and single-cell data generated on the same mass analyzers. Instrument acquisition files, processed data, and spectrum libraries are publicly available on MASSIVE via accession MSV000090635.

Achieving a Deeper Understanding of Drug Metabolism and Responses Using Single-Cell Technologies.

Recent advancements in single-cell technologies have enabled detection of RNA, proteins, metabolites, and xenobiotics in individual cells, and the application of these technologies has the potential to transform pharmacological research. Single-cell data has already resulted in the development of human and model species cell atlases, identifying different cell types within a tissue, further facilitating the characterization of tumor heterogeneity, and providing insight into treatment resistance. Research discussed in this review demonstrates that distinct cell populations express drug metabolizing enzymes to different extents, indicating there may be variability in drug metabolism not only between organs, but within tissue types. Additionally, we put forth the concept that single-cell analyses can be used to expose underlying variability in cellular response to drugs, providing a unique examination of drug efficacy, toxicity, and metabolism. We will outline several of these techniques: single-cell RNA-sequencing and mass cytometry to characterize and distinguish different cell types, single-cell proteomics to quantify drug metabolizing enzymes and characterize cellular responses to drug, capillary electrophoresis-ultrasensitive laser-induced fluorescence detection and single-probe single-cell mass spectrometry for detection of drugs, and others. Emerging single-cell technologies such as these can comprehensively characterize heterogeneity in both cell-type-specific drug metabolism and response to treatment, enhancing progress toward personalized and precision medicine. SIGNIFICANCE STATEMENT: Recent technological advances have enabled the analysis of gene expression and protein levels in single cells. These types of analyses are important to investigating mechanisms that cannot be elucidated on a bulk level, primarily due to the variability of cell populations within biological systems. Here, we summarize cell-type-specific drug metabolism and how pharmacologists can utilize single-cell approaches to obtain a comprehensive understanding of drug metabolism and cellular heterogeneity in response to drugs.

EstG is a novel esterase required for cell envelope integrity in Caulobacter.

Proper regulation of the bacterial cell envelope is critical for cell survival. Identification and characterization of enzymes that maintain cell envelope homeostasis is crucial, as they can be targets for effective antibiotics. In this study, we have identified a novel enzyme, called EstG, whose activity protects cells from a variety of lethal assaults in the ⍺-proteobacterium Caulobacter crescentus. Despite homology to transpeptidase family cell wall enzymes and an ability to protect against cell-wall-targeting antibiotics, EstG does not demonstrate biochemical activity toward cell wall substrates. Instead, EstG is genetically connected to the periplasmic enzymes OpgH and BglX, responsible for synthesis and hydrolysis of osmoregulated periplasmic glucans (OPGs), respectively. The crystal structure of EstG revealed similarities to esterases and transesterases, and we demonstrated esterase activity of EstG in vitro. Using biochemical fractionation, we identified a cyclic hexamer of glucose as a likely substrate of EstG. This molecule is the first OPG described in Caulobacter and establishes a novel class of OPGs, the regulation and modification of which are important for stress survival and adaptation to fluctuating environments. Our data indicate that EstG, BglX, and OpgH comprise a previously unknown OPG pathway in Caulobacter. Ultimately, we propose that EstG is a novel enzyme that instead of acting on the cell wall, acts on cyclic OPGs to provide resistance to a variety of cellular stresses.

Insights into protein post-translational modification landscapes of individual human cells by trapped ion mobility time-of-flight mass spectrometry.

Single cell proteomics is a powerful tool with potential for markedly enhancing understanding of cellular processes. Here we report the development and application of multiplexed single cell proteomics using trapped ion mobility time-of-flight mass spectrometry. When employing a carrier channel to improve peptide signal, this method allows over 40,000 tandem mass spectra to be acquired in 30 min. Using a KRASG12C model human-derived cell line, we demonstrate the quantification of over 1200 proteins per cell with high relative sequence coverage permitting the detection of multiple classes of post-translational modifications in single cells. When cells were treated with a KRASG12C covalent inhibitor, this approach revealed cell-to-cell variability in the impact of the drug, providing insight missed by traditional proteomics. We provide multiple resources necessary for the application of single cell proteomics to drug treatment studies including tools to reduce cell cycle linked proteomic effects from masking pharmacological phenotypes.