Diversity of post-translational modifications and cell signaling revealed by single cell and single organelle mass spectrometry.

The rapid evolution of mass spectrometry-based single-cell proteomics now enables the cataloging of several thousand proteins from single cells. We investigated whether we could discover cellular heterogeneity beyond proteome, encompassing post-translational modifications (PTM), protein-protein interaction, and variants. By optimizing the mass spectrometry data interpretation strategy to enable the detection of PTMs and variants, we have generated a high-definition dataset of single-cell and nuclear proteomic-states. The data demonstrate the heterogeneity of cell-states and signaling dependencies at the single-cell level and reveal epigenetic drug-induced changes in single nuclei. This approach enables the exploration of previously uncharted single-cell and organellar proteomes revealing molecular characteristics that are inaccessible through RNA profiling.

Exploration of Nitrotyrosine-Containing Proteins and Peptides by Antibody-Based Enrichment Strategies.

Nitrotyrosine, or 3-nitrotyrosine, is an oxidative post-translational modification induced by reactive nitrogen species. Although nitrotyrosine is considered a marker of oxidative stress and has been associated with inflammation, neurodegeneration, cardiovascular disease, and cancer, identification of nitrotyrosine-modified proteins remains challenging owing to its low stoichiometric levels in biological samples. To facilitate a comprehensive analysis of proteins and peptides containing nitrotyrosine, we optimized an immunoprecipitation-based enrichment workflow using a cell line model. The identification of proteins and peptides containing nitrotyrosine residues was carried out after peroxynitrite treatment of cell lysates, which generated modified nitrotyrosine residues on susceptible sites on proteins. We evaluated the efficacy of enriching nitrotyrosine-modified proteins and peptides by employing four different commercially available monoclonal antibodies directed against nitrotyrosine. LC-MS/MS analysis resulted in the identification of 1377 and 1624 nitrotyrosine-containing peptides from protein- and peptide-based enrichment experiments, respectively. Although the yield of nitrotyrosine-containing peptides was higher in experiments where peptides rather than proteins were enriched, we found a substantial proportion (37-65%) of identified nitrotyrosine-containing peptides contained nitrotyrosine at the N-terminus. However, in protein-based immunoprecipitation <9% of nitrotyrosine-containing peptides had nitrotyrosine modification at the N-terminus of the peptide. Overall, our study resulted in the identification of 2603 nitrotyrosine-containing peptides of which >2000 have not previously been reported. We synthesized 101 novel nitrotyrosine-containing peptides identified in our analysis and analyzed them by LC-MS/MS to validate our findings. We have confirmed the validity of 70% of these peptides, as they demonstrated a similarity score exceeding 0.7 when compared to peptides identified through experimental methods. Finally, we also validated the presence of nitrotyrosine modification on PKM and EF2 proteins in peroxynitrite-treated samples by immunoblot analysis. The large catalog presented in this study along with the workflow should facilitate the investigation of nitrotyrosine as an oxidative modification in a variety of settings in greater detail.

13C15N: glucagon-based novel isotope dilution mass spectrometry method for measurement of glucagon metabolism in humans.

BACKGROUND: Glucagon serves as an important regulatory hormone for regulating blood glucose concentration with tight feedback control exerted by insulin and glucose. There are critical gaps in our understanding of glucagon kinetics, pancreatic α cell function and intra-islet feedback network that are disrupted in type 1 diabetes. This is important for translational research applications of evolving dual-hormone (insulin + glucagon) closed-loop artificial pancreas algorithms and their usage in type 1 diabetes. Thus, it is important to accurately measure glucagon kinetics in vivo and to develop robust models of glucose-insulin-glucagon interplay that could inform next generation of artificial pancreas algorithms. METHODS: Here, we describe the administration of novel 13C15N heavy isotope-containing glucagon tracers-FF glucagon [(Phe 6 13C9,15N; Phe 22 13C9,15N)] and FFLA glucagon [(Phe 6 13C9,15N; Phe 22 13C9,15N; Leu 14 13C6,15N; Ala 19 13C3)] followed by anti-glucagon antibody-based enrichment and LC-MS/MS based-targeted assays using high-resolution mass spectrometry to determine levels of infused glucagon in plasma samples. The optimized assay results were applied for measurement of glucagon turnover in subjects with and without type 1 diabetes infused with isotopically labeled glucagon tracers. RESULTS: The limit of quantitation was found to be 1.56 pg/ml using stable isotope-labeled glucagon as an internal standard. Intra and inter-assay variability was < 6% and < 16%, respectively, for FF glucagon while it was < 5% and < 23%, respectively, for FFLA glucagon. Further, we carried out a novel isotope dilution technique using glucagon tracers for studying glucagon kinetics in type 1 diabetes. CONCLUSIONS: The methods described in this study for simultaneous detection and quantitation of glucagon tracers have clinical utility for investigating glucagon kinetics in vivo in humans.

Self-Care for Advanced Practice Nursing Students in Rural Primary Care.

BACKGROUND: Advanced practice RNs (APRNs) working in rural primary care provide a spectrum of health care needs that can lead to professional burnout. As a preventative for burnout, the Institute of Medicine developed a strategy focused on self-care. Understanding the importance of self-care as a preventive for building professional resilience to manage workplace stressors during students' academic years may improve retention. METHOD: Twenty-two APRN students participated in a rural primary care immersion course with a specific component on self-care. Preand postsurveys, student journaling, class discussions, and a posttraining focus group were used to assess students' progress. RESULTS: Students reported strengthening patient and team relationships, enhanced interpersonal communication, and increased self-awareness of stressors for managing their emotions. These self-imposed processes improved student confidence, job satisfaction, and workplace resilience. CONCLUSION: Heath-promoting behaviors via self-care during APRN students' formal education may assist them in their clinical practice as rural primary care providers. [J Nurs Educ. 2022;61(4):187-191.].

DIA-Based Proteome Profiling of Nasopharyngeal Swabs from COVID-19 Patients.

Since the recent outbreak of COVID-19, there have been intense efforts to understand viral pathogenesis and host immune response to combat SARS-CoV-2. It has become evident that different host alterations can be identified in SARS-CoV-2 infection based on whether infected cells, animal models or clinical samples are studied. Although nasopharyngeal swabs are routinely collected for SARS-CoV-2 detection by RT-PCR testing, host alterations in the nasopharynx at the proteomic level have not been systematically investigated. Thus, we sought to characterize the host response through global proteome profiling of nasopharyngeal swab specimens. A mass spectrometer combining trapped ion mobility spectrometry (TIMS) and high-resolution QTOF mass spectrometer with parallel accumulation-serial fragmentation (PASEF) was deployed for unbiased proteome profiling. First, deep proteome profiling of pooled nasopharyngeal swab samples was performed in the PASEF enabled DDA mode, which identified 7723 proteins that were then used to generate a spectral library. This approach provided peptide level evidence of five missing proteins for which MS/MS spectrum and mobilograms were validated with synthetic peptides. Subsequently, quantitative proteomic profiling was carried out for 90 individual nasopharyngeal swab samples (45 positive and 45 negative) in DIA combined with PASEF, termed as diaPASEF mode, which resulted in a total of 5023 protein identifications. Of these, 577 proteins were found to be upregulated in SARS-CoV-2 positive samples. Functional analysis of these upregulated proteins revealed alterations in several biological processes including innate immune response, viral protein assembly, and exocytosis. To the best of our knowledge, this study is the first to deploy diaPASEF for quantitative proteomic profiling of clinical samples and shows the feasibility of adopting such an approach to understand mechanisms and pathways altered in diseases.

A mass spectrometry-based targeted assay for detection of SARS-CoV-2 antigen from clinical specimens.

BACKGROUND: The COVID-19 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has overwhelmed health systems worldwide and highlighted limitations of diagnostic testing. Several types of diagnostic tests including RT-PCR-based assays and antigen detection by lateral flow assays, each with their own strengths and weaknesses, have been developed and deployed in a short time. METHODS: Here, we describe an immunoaffinity purification approach followed a by high resolution mass spectrometry-based targeted qualitative assay capable of detecting SARS-CoV-2 viral antigen from nasopharyngeal swab samples. Based on our discovery experiments using purified virus, recombinant viral protein and nasopharyngeal swab samples from COVID-19 positive patients, nucleocapsid protein was selected as a target antigen. We then developed an automated antibody capture-based workflow coupled to targeted high-field asymmetric waveform ion mobility spectrometry (FAIMS) - parallel reaction monitoring (PRM) assay on an Orbitrap Exploris 480 mass spectrometer. An ensemble machine learning-based model for determining COVID-19 positive samples was developed using fragment ion intensities from the PRM data. FINDINGS: The optimized targeted assay, which was used to analyze 88 positive and 88 negative nasopharyngeal swab samples for validation, resulted in 98% (95% CI = 0.922-0.997) (86/88) sensitivity and 100% (95% CI = 0.958-1.000) (88/88) specificity using RT-PCR-based molecular testing as the reference method. INTERPRETATION: Our results demonstrate that direct detection of infectious agents from clinical samples by tandem mass spectrometry-based assays have potential to be deployed as diagnostic assays in clinical laboratories, which has hitherto been limited to analysis of pure microbial cultures. FUNDING: This study was supported by DBT/Wellcome Trust India Alliance Margdarshi Fellowship grant IA/M/15/1/502023 awarded to AP and the generosity of Eric and Wendy Schmidt.

Digging deeper into the immunopeptidome: characterization of post-translationally modified peptides presented by MHC I.

Peptides presented by MHC molecules on the cell surface, or the immunopeptidome, play an important role in the adaptive arm of the immune response. Antigen processing for MHC class I molecules is a ubiquitous pathway present in all nucleated cells which generates and presents peptides of both self and non-self-origin. Peptides with post-translational modifications represent one category of peptides presented by MHC class I molecules. However, owing to the complexity of self-peptides presented by cells, the diversity of peptides with post-translational modifications is not well-studied. In this study, we carried out MHC Class I immunopeptidomics analysis of Loucy T-cell leukemia and A375 malignant melanoma cell line to characterize the diversity of post-translational modifications of MHC class I-bound peptides. Using high resolution mass spectrometry, we identified 25,761 MHC-bound peptides across both cell lines using Bolt and Sequest search engines. The enrichment method was highly specific as ~ 90% of the peptides were of typical length (8-12 amino acids long) and the motifs were expected based on previously reported motifs for MHC I alleles. Among the MHC-bound peptides, we identified phosphorylation as a major post-translational modification followed by deamidation. We observed site-specific localization of these post-translational modifications, at position P4 for phosphorylated peptides and position P3 for deamidated peptides. We identified a smaller number of peptides with acetylated and methylated lysine, possibly due to very low stoichiometric levels of these PTMs compared to phosphorylation and deamidation. Using PEAKS de novo sequencing algorithm, we identified spliced peptides that accounted for ~ 5-7% of MHC-bound peptides that were otherwise similar in their features as normal MHC-bound peptides. We validated the identity of several post-translationally modified peptides and spliced peptides through mass spectrometric analysis of synthetic peptides. Our study confirms post-translationally modified peptides to be present at low stoichiometric levels along with unusual spliced peptides through unbiased identification using high resolution mass spectrometry. Supplementary Information: The online version contains supplementary material available at 10.1007/s42485-021-00066-x.

Preparing Nurse Practitioner Students to Practice in Rural Primary Care.

BACKGROUND: Gaps remain in rural primary care. To fill this gap, nurse practioner (NP) graduates may need additional training using a rural-specific curriculum framework to be ready to practice in rural primary care. METHOD: Ten NP students participated in a 16-week rural immersion. Preand postsurveys, online journaling, self-guided testing, simulation events, and postcourse focus groups were used to evaluate student progress using directed content analysis to identify key themes and to verify, organize, and categorize the collected data. RESULTS: Students reported gains in rural culture competence, increased skills in health literacy and patient advocacy, improved communication and negotiating ethical issues with patients, and increased awareness of challenges in rural health care and the importance of resilience. CONCLUSION: An immersion learning experience with targeted didactic content and clinical practicum in rural primary care can help to enhance NP students' confidence and technical abilities for providing optimal rural primary care. [J Nurs Educ. 2020;59(10):581-584.].

Development of an instrument to assess nurse practitioner attitudes and beliefs about obesity.

BACKGROUND AND PURPOSE: Obesity has been identified as the number one health problem in the United States. Estimates are that almost 70% of adults are overweight or obese. The care received by these individuals may be influenced by their weight status. The purpose of this study was to describe the self-reported attitudes and beliefs of nurse practitioners (NPs) toward this population. METHODS: Data were obtained from 358 NPs who were attending a national conference. Each participant completed the investigator-developed survey, which assessed attitudes and beliefs, and provided limited demographic information so that the study population could be described. CONCLUSIONS: Scores indicate that these NPs perceived overweight and obese persons to be (1) not as good as others, (2) not as successful as others, (3) not suitable for marriage, (4) untidy, and (5) not as healthy. IMPLICATIONS FOR PRACTICE: While bias exists with respect to overweight and obese persons, awareness and knowledge of one's personal bias is the first step in preventing this perception to influence care. Educational forums should include a personal assessment and interventions aimed at minimizing the effect one's attitude and belief has on the delivery of respectful care.

The Role and Influence of Faith Leaders on Health-Related Issues and Programs in their Congregation.

This qualitative study explored the influence of faith leaders on health-related issues within their congregation. Semi-structured interviewers with 24 faith leaders found that chronic conditions and poor health behaviors were the top health challenges facing their congregation. A majority mentioned health-related activities taking place at their church. Most believed they had influence on their congregation for issues related to health/wellness, most commonly in the form of increasing awareness. A majority talked about the importance of being a role model. It is important to understand how to most effectively capitalize on the strengths of and engage pastors in health promotion efforts.

Leading their flocks to health? Clergy health and the role of clergy in faith-based health promotion interventions.

Faith-based organizations are a frequent partner in health promotion due to their large and expansive reach across multiple demographics of the United States. These faith-based organizations are led by clergy members who have a strong influence over their institutions and who shape the physical and social environments of their institutions for health-related matters. The purpose of this review was to examine current issues associated with the health, behaviors, and well-being of clergy, highlight the literature on the role clergy play in delivering effective health promotion interventions, and present recommendations for improving clergy health and the involvement of clergy in faith-based initiatives.

Psychometric evaluation of the Shared Decision-Making Instrument--Revised.

The purpose of this study was to evaluate the psychometric properties of the Shared Decision-Making Inventory-Revised (SDMI-R) to measure four constructs (knowledge, attitudes, self-efficacy, and intent) theoretically defined as vital in discussing the human papillomavirus (HPV) disease and vaccine with clients. The SDMI-R was distributed to a sample (N = 1,525) of school nurses. Correlational matrixes denoted moderate to strong correlations, indicating adequate internal reliability. Reliability for the total instrument was satisfactory (α = .874) along with Attitude, Self-Efficacy and Intent subscales .828, .917, .891, respectively. Exploratory factor analysis revealed five components that explained 75.96% of the variance.

Heart and soul physical activity program for African American women.

African American women are less physically active than White women and have a higher prevalence of inactivity-related diseases. Increased physical activity is known to reduce the risks for many chronic diseases. Positive health behavior changes have resulted from health promotion interventions conducted in African American churches. Eighteen midlife African American women participated in the Heart and Soul Physical Activity Program (HSPAP), a church-based physical activity intervention conceptualized in appraisal, belonging, tangible, and self-esteem domains of social support. Feasibility of the HSPAP was tested by determining changes in time and intensity of physical activity and social support for physical activity from baseline to 6 weeks. Data analyses were conducted utilizing paired t tests. Significant increases in time spent in physical activity from a mean of 412 min/week at baseline to 552 min/week at 6 weeks were noted. Participants reported increases in social support for physical activity in the 6-week study.

Design and chemical synthesis of a magnetic resonance contrast agent with enhanced in vitro binding, high blood-brain barrier permeability, and in vivo targeting to Alzheimer's disease amyloid plaques.

Molecular imaging is an important new direction in medical diagnosis; however, its success is dependent upon molecular probes that demonstrate selective tissue targeting. We report the design and chemical synthesis of a derivative of human amyloid-beta (Abeta) peptide that is capable of selectively targeting individual amyloid plaques in the brain of Alzheimer's disease transgenic mice after being intravenously injected. This derivative is based on the sequence of the first 30 amino acid residues of Abeta with asparagyl/glutamyl-4-aminobutane residues (N-4ab/Q-4ab) substituted at unique Asp and Glu positions and with Gd-DTPA-aminohexanoic acid covalently attached at the N-terminal Asp. The Gd[N-4ab/Q-4ab]Abeta30 peptide was homogeneous as shown by high-resolution analytical techniques with a mass of +/-4385 Da determined by electrospray ionization mass spectrometry. This diamine- and gadolinium-substituted derivative of Abeta is shown to have enhanced in vitro binding to Alzheimer's disease (AD) amyloid plaques and increased in vivo permeability at the blood-brain barrier because of the unique Asp/Glu substitutions. In addition, specific in vivo targeting to AD amyloid plaques is demonstrated throughout the brain of an APP, PS1 transgenic mouse after intravenous injection. Because of the magnetic resonance (MR) imaging contrast enhancement provided by gadolinium, this derivative should enable the in vivo MR imaging of individual amyloid plaques in the brains of AD animals or patients to allow for early diagnosis and also provide a direct measure of the efficacy of anti-amyloid therapies currently being developed.