A Multipathway Phosphopeptide Standard for Rapid Phosphoproteomics Assay Development.

Recent advances in methodology have made phosphopeptide analysis a tractable problem for many proteomics researchers. There are now a wide variety of robust and accessible enrichment strategies to generate phosphoproteomes while free or inexpensive software tools for quantitation and site localization have simplified phosphoproteome analysis workflow tremendously. As a research group under the Association for Biomolecular Resource Facilities umbrella, the Proteomics Standards Research Group has worked to develop a multipathway phosphopeptide standard based on a mixture of heavy-labeled phosphopeptides designed to enable researchers to rapidly develop assays. This mixture contains 131 mass spectrometry vetted phosphopeptides specifically chosen to cover as many known biologically interesting phosphosites as possible from seven different signaling networks: AMPK signaling, death and apoptosis signaling, ErbB signaling, insulin/insulin-like growth factor-1 signaling, mTOR signaling, PI3K/AKT signaling, and stress (p38/SAPK/JNK) signaling. Here, we describe a characterization of this mixture spiked into a HeLa tryptic digest stimulated with both epidermal growth factor and insulin-like growth factor-1 to activate the MAPK and PI3K/AKT/mTOR pathways. We further demonstrate a comparison of phosphoproteomic profiling of HeLa performed independently in five labs using this phosphopeptide mixture with data-independent acquisition. Despite different experimental and instrumentation processes, we found that labs could produce reproducible, harmonized datasets by reporting measurements as ratios to the standard, while intensity measurements showed lower consistency between labs even after normalization. Our results suggest that widely available, biologically relevant phosphopeptide standards can act as a quantitative "yardstick" across laboratories and sample preparations enabling experimental designs larger than a single laboratory can perform. Raw data files are publicly available in the MassIVE dataset MSV000090564.

The Performance of an Artificial Neural Network Model in Predicting the Early Distribution Kinetics of Propofol in Morbidly Obese and Lean Subjects.

BACKGROUND: Induction of anesthesia is a phase characterized by rapid changes in both drug concentration and drug effect. Conventional mammillary compartmental models are limited in their ability to accurately describe the early drug distribution kinetics. Recirculatory models have been used to account for intravascular mixing after drug administration. However, these models themselves may be prone to misspecification. Artificial neural networks offer an advantage in that they are flexible and not limited to a specific structure and, therefore, may be superior in modeling complex nonlinear systems. They have been used successfully in the past to model steady-state or near steady-state kinetics, but never have they been used to model induction-phase kinetics using a high-resolution pharmacokinetic dataset. This study is the first to use an artificial neural network to model early- and late-phase kinetics of a drug. METHODS: Twenty morbidly obese and 10 lean subjects were each administered propofol for induction of anesthesia at a rate of 100 mg/kg/h based on lean body weight and total body weight for obese and lean subjects, respectively. High-resolution plasma samples were collected during the induction phase of anesthesia, with the last sample taken at 16 hours after propofol administration for a total of 47 samples per subject. Traditional mammillary compartment models, recirculatory models, and a gated recurrent unit neural network were constructed to model the propofol pharmacokinetics. Model performance was compared. RESULTS: A 4-compartment model, a recirculatory model, and a gated recurrent unit neural network were assessed. The final recirculatory model (mean prediction error: 0.348; mean square error: 23.92) and gated recurrent unit neural network that incorporated ensemble learning (mean prediction error: 0.161; mean square error: 20.83) had similar performance. Each of these models overpredicted propofol concentrations during the induction and elimination phases. Both models had superior performance compared to the 4-compartment model (mean prediction error: 0.108; mean square error: 31.61), which suffered from overprediction bias during the first 5 minutes followed by under-prediction bias after 5 minutes. CONCLUSIONS: A recirculatory model and gated recurrent unit artificial neural network that incorporated ensemble learning both had similar performance and were both superior to a compartmental model in describing our high-resolution pharmacokinetic data of propofol. The potential of neural networks in pharmacokinetic modeling is encouraging but may be limited by the amount of training data available for these models.

Enhancing Data Reliability in TOMAHAQ for Large-Scale Protein Quantification.

The analytical scale of most mass-spectrometry-based targeted proteomics assays is usually limited by assay performance and instrument utilization. A recently introduced method, called triggered by offset, multiplexed, accurate mass, high resolution, and absolute quantitation (TOMAHAQ), combines both peptide and sample multiplexing to simultaneously improve analytical scale and quantitative performance. In the present work, critical technical requirements and data analysis considerations for successful implementation of the TOMAHAQ technique based on the study of a total of 185 target peptides across over 200 clinical plasma samples are discussed. Importantly, it is observed that significant interference originate from the TMTzero reporter ion used for the synthetic trigger peptides. This interference is not expected because only TMT10plex reporter ions from the target peptides should be observed under typical TOMAHAQ conditions. In order to unlock the great promise of the technique for high throughput quantification, here a post-acquisition data correction strategy to deconvolute the reporter ion superposition and recover reliable data is proposed.

Identification of Widespread Antibiotic Exposure in Patients With Cholera Correlates With Clinically Relevant Microbiota Changes.

BACKGROUND: A first step to combating antimicrobial resistance in enteric pathogens is to establish an objective assessment of antibiotic exposure. Our goal was to develop and evaluate a liquid chromatography-ion trap mass spectrometry (LC/MS) method to determine antibiotic exposure in patients with cholera. METHODS: A priority list for targeted LC/MS was generated from medication-vendor surveys in Bangladesh. A study of patients with and those without cholera was conducted to collect and analyze paired urine and stool samples. RESULTS: Among 845 patients, 11% (90) were Vibrio cholerae positive; among these 90 patients, analysis of stool specimens revealed ≥1 antibiotic in 86% and ≥2 antibiotics in 52%. Among 44 patients with cholera and paired urine and stool specimens, ≥1 antibiotic was detected in 98% and ≥2 antibiotics were detected in 84%, despite 55% self-reporting medication use. Compared with LC/MS, a low-cost antimicrobial detection bioassay lacked a sufficient negative predictive value (10%; 95% confidence interval, 6%-16%). Detection of guideline-recommended antibiotics in stool specimens did (for azithromycin; P = .040) and did not (for ciprofloxacin) correlate with V. cholerae suppression. A nonrecommended antibiotic (metronidazole) was associated with decreases in anaerobes (ie, Prevotella organisms; P < .001). CONCLUSION: These findings suggest that there may be no true negative control group when attempting to account for antibiotic exposure in settings like those in this study.

Plasma anandamide concentrations are lower in children with autism spectrum disorder.

Background: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by restricted, stereotyped behaviors and impairments in social communication. Although the underlying biological mechanisms of ASD remain poorly understood, recent preclinical research has implicated the endogenous cannabinoid (or endocannabinoid), anandamide, as a significant neuromodulator in rodent models of ASD. Despite this promising preclinical evidence, no clinical studies to date have tested whether endocannabinoids are dysregulated in individuals with ASD. Here, we addressed this critical gap in knowledge by optimizing liquid chromatography-tandem mass spectrometry methodology to quantitatively analyze anandamide concentrations in banked blood samples collected from a cohort of children with and without ASD (N = 112). Findings: Anandamide concentrations significantly differentiated ASD cases (N = 59) from controls (N = 53), such that children with lower anandamide concentrations were more likely to have ASD (p = 0.041). In keeping with this notion, anandamide concentrations were also significantly lower in ASD compared to control children (p = 0.034). Conclusions: These findings are the first empirical human data to translate preclinical rodent findings to confirm a link between plasma anandamide concentrations in children with ASD. Although preliminary, these data suggest that impaired anandamide signaling may be involved in the pathophysiology of ASD.

Proteomics of Primary Cilia by Proximity Labeling.

While cilia are recognized as important signaling organelles, the extent of ciliary functions remains unknown because of difficulties in cataloguing proteins from mammalian primary cilia. We present a method that readily captures rapid snapshots of the ciliary proteome by selectively biotinylating ciliary proteins using a cilia-targeted proximity labeling enzyme (cilia-APEX). Besides identifying known ciliary proteins, cilia-APEX uncovered several ciliary signaling molecules. The kinases PKA, AMPK, and LKB1 were validated as bona fide ciliary proteins and PKA was found to regulate Hedgehog signaling in primary cilia. Furthermore, proteomics profiling of Ift27/Bbs19 mutant cilia correctly detected BBSome accumulation inside Ift27(-/-) cilia and revealed that ß-arrestin 2 and the viral receptor CAR are candidate cargoes of the BBSome. This work demonstrates that proximity labeling can be applied to proteomics of non-membrane-enclosed organelles and suggests that proteomics profiling of cilia will enable a rapid and powerful characterization of ciliopathies.

Neural precursor cell-expressed developmentally down-regulated protein 4-2 (Nedd4-2) regulation by 14-3-3 protein binding at canonical serum and glucocorticoid kinase 1 (SGK1) phosphorylation sites.

Regulation of epithelial Na(+) channel (ENaC)-mediated transport in the distal nephron is a critical determinant of blood pressure in humans. Aldosterone via serum and glucocorticoid kinase 1 (SGK1) stimulates ENaC by phosphorylation of the E3 ubiquitin ligase Nedd4-2, which induces interaction with 14-3-3 proteins. However, the mechanisms of SGK1- and 14-3-3-mediated regulation of Nedd4-2 are unclear. There are three canonical SGK1 target sites on Nedd4-2 that overlap phosphorylation-dependent 14-3-3 interaction motifs. Two of these are termed "minor," and one is termed "major," based on weak or strong binding to 14-3-3 proteins, respectively. By mass spectrometry, we found that aldosterone significantly stimulates phosphorylation of a minor, relative to the major, 14-3-3 binding site on Nedd4-2. Phosphorylation-deficient minor site Nedd4-2 mutants bound less 14-3-3 than did wild-type (WT) Nedd4-2, and minor site Nedd4-2 mutations were sufficient to inhibit SGK1 stimulation of ENaC cell surface expression. As measured by pulse-chase and cycloheximide chase assays, a major binding site Nedd4-2 mutant had a shorter cellular half-life than WT Nedd4-2, but this property was not dependent on binding to 14-3-3. Additionally, a dimerization-deficient 14-3-3ε mutant failed to bind Nedd4-2. We conclude that whereas phosphorylation at the Nedd4-2 major site is important for interaction with 14-3-3 dimers, minor site phosphorylation by SGK1 may be the relevant molecular switch that stabilizes Nedd4-2 interaction with 14-3-3 and thus promotes ENaC cell surface expression. We also propose that major site phosphorylation promotes cellular Nedd4-2 protein stability, which potentially represents a novel form of regulation for turnover of E3 ubiquitin ligases.

The ABRF Proteomics Research Group studies: educational exercises for qualitative and quantitative proteomic analyses.

Resource (core) facilities have played an ever-increasing role in furnishing the scientific community with specialized instrumentation and expertise for proteomics experiments in a cost-effective manner. The Proteomics Research Group (PRG) of the Association of Biomolecular Resource Facilities (ABRF) has sponsored a number of research studies designed to enable participants to try new techniques and assess their capabilities relative to other laboratories analyzing the same samples. Presented here are results from three PRG studies representing different samples that are typically analyzed in a core facility, ranging from simple protein identification to targeted analyses, and include intentional challenges to reflect realistic studies. The PRG2008 study compares different strategies for the qualitative characterization of proteins, particularly the utility of complementary methods for characterizing truncated protein forms. The use of different approaches for determining quantitative differences for several target proteins in human plasma was the focus of the PRG2009 study. The PRG2010 study explored different methods for determining specific constituents while identifying unforeseen problems that could account for unanticipated results associated with the different samples, and included (15) N-labeled proteins as an additional challenge. These studies provide a valuable educational resource to research laboratories and core facilities, as well as a mechanism for establishing good laboratory practices.

Azetidine-2-carboxylic acid in the food chain.

Azetidine-2-carboxylic acid (Aze) 1 is a non-protein amino acid present in sugar beets and in table beets (Beta vulgaris). It is readily misincorporated into proteins in place of proline 2 in many species, including humans, and causes numerous toxic effects as well as congenital malformations. Its role in the pathogenesis of disease in humans has remained unexplored. Sugar beet agriculture, especially in the Northern Hemisphere, has become widespread during the past 150 years, and now accounts for nearly 30% of the world's supply of sucrose. Sugar beet byproducts are also used as a dietary supplement for livestock. Therefore, this study was undertaken as an initial survey to identify Aze-containing links in the food chain. Herein, we report the presence of Aze 1 in three sugar beet byproducts that are fed to farm animals: sugar beet molasses, shredded sugar beet pulp, and pelleted sugar beet pulp.

Azetidine-2-carboxylic acid in garden beets (Beta vulgaris).

Azetidine-2-carboxylic acid (L-Aze) is a toxic and teratogenic non-protein amino acid. In many species, including man, L-Aze is misincorporated into protein in place of proline, altering collagen, keratin, hemoglobin, and protein folding. In animal models of teratogenesis, it causes a wide range of malformations. The role of L-Aze in human disease has been unexplored, probably because the compound has not been associated with foods consumed by humans. Herein we report the presence of L-Aze in the garden or table beet (Beta vulgaris).

A double-vented tetraphasic continuous column approach to MuDPIT analysis on long capillary columns demonstrates superior proteomic coverage.

A double-vented serial tetraphasic capillary column approach is applied to proteomic MuDPIT-type analysis using extended length capillary reverse-phase columns. The heart of the tetraphasic device consists of a triphasic MuDPIT trap located upstream of a venting tee. The trap is followed by a 60 cm high-resolution capillary column. A conventional high-flow HPLC is used to develop gradients at standard flow rates and pressures. The double-vented triphasic MuDPIT trapping device relieves the capillary separation column from the salt burden during the on-line cation-exchange portion of the analysis. Two configurations are presented, a double-vented continuous column model and a discontinuous model in which the triphasic MuDPIT trap is installed on a six-port valve; both configurations were tested with 60 and 10 cm capillary columns. All four systems were challenged with a trypsin digest of undepleted human serum, and a matrix of proteomic results for the different models and column lengths are compared.