A crustacean neuropeptide spectral library for data-independent acquisition (DIA) mass spectrometry applications.

Neuropeptides have tremendous potential for application in modern medicine, including utility as biomarkers and therapeutics. To overcome the inherent challenges associated with neuropeptide identification and characterization, data-independent acquisition (DIA) is a fitting mass spectrometry (MS) method of choice to achieve sensitive and accurate analysis. It is advantageous for preliminary neuropeptidomic studies to occur in less complex organisms, with crustacean models serving as a popular choice due to their relatively simple nervous system. With spectral libraries serving as a means to interpret DIA-MS output spectra, and Cancer borealis as a model of choice for neuropeptide analysis, we performed the first spectral library mapping of crustacean neuropeptides. Leveraging pre-existing data-dependent acquisition (DDA) spectra, a spectral library was built using PEAKS Online. The library is comprised of 333 unique neuropeptides. The identification results obtained through the use of this spectral library were compared with those achieved through library-free analysis of crustacean brain, pericardial organs (PO), and thoracic ganglia (TG) tissues. A statistically significant increase (Student's t-test, P value < 0.05) in the number of identifications achieved from the TG data was observed in the spectral library results. Furthermore, in each of the tissues, a distinctly different set of identifications was found in the library search compared to the library-free search. This work highlights the necessity for the use of spectral libraries in neuropeptide analysis, illustrating the advantage of spectral libraries for interpreting DIA spectra in a reproducible manner with greater neuropeptidomic depth.

EndoGenius: Optimized Neuropeptide Identification from Mass Spectrometry Datasets.

Neuropeptides represent a unique class of signaling molecules that have garnered much attention but require special consideration when identifications are gleaned from mass spectra. With highly variable sequence lengths, neuropeptides must be analyzed in their endogenous state. Further, neuropeptides share great homology within families, differing by as little as a single amino acid residue, complicating even routine analyses and necessitating optimized computational strategies for confident and accurate identifications. We present EndoGenius, a database searching strategy designed specifically for elucidating neuropeptide identifications from mass spectra by leveraging optimized peptide-spectrum matching approaches, an expansive motif database, and a novel scoring algorithm to achieve broader representation of the neuropeptidome and minimize reidentification. This work describes an algorithm capable of reporting more neuropeptide identifications at 1% false-discovery rate than alternative software in five Callinectes sapidus neuronal tissue types.

Mass Spectrometry-Based Precise Identification of Citrullinated Histones via Limited Digestion and Biotin Derivative Tag Enrichment.

Histone citrullination is an essential epigenetic post-translational modification (PTM) that affects many important physiological and pathological processes, but effective tools to study histone citrullination are greatly limited due to several challenges, including the small mass shift caused by this PTM and its low abundance in biological systems. Although previous studies have reported frequent occurrences of histone citrullination, these methods failed to provide a high-throughput and site-specific strategy to detect histone citrullination. Recently, we developed a biotin thiol tag that enabled precise identification of protein citrullination coupled with mass spectrometry. However, very few histone citrullination sites were identified, likely due to the highly basic nature of these proteins. In this study, we develop a novel method utilizing limited digestion and biotin derivative tag enrichment to facilitate direct in vivo identification of citrullination sites on histones. We achieve improved coverage of histone identification via partial enzymatic digestion and lysine block by dimethylation. With biotin tag-assisted chemical derivatization and enrichment, we also achieve precise annotation of histone citrullination sites with high confidence. We further compare different fragmentation methods and find that the electron-transfer-dissociation-based approach enables the most in-depth analysis and characterization. In total, we unambiguously identify 18 unique citrullination sites on histones in human astrocytoma U87 cells, including 15 citrullinated sites being detected for the first time. Some of these citrullination sites are observed to exhibit noticeable alterations in response to DNA damage, which demonstrates the superiority of our strategy in understanding the roles of histone citrullination in critical biological processes.

Recent advances in characterization of citrullination and its implication in human disease research: From method development to network integration.

Post-translational modifications (PTM) of proteins increase the functional diversity of the proteome and have been implicated in the pathogenesis of numerous diseases. The most widely understood modifications include phosphorylation, methylation, acetylation, O-linked/N-linked glycosylation, and ubiquitination, all of which have been extensively studied and documented. Citrullination is a historically less explored, yet increasingly studied, protein PTM which has profound effects on protein conformation and protein-protein interactions. Dysregulation of protein citrullination has been associated with disease development and progression. Identification and characterization of citrullinated proteins is highly challenging, complicated by the low cellular abundance of citrullinated proteins, making it difficult to identify and quantify the extent of citrullination in samples, coupled with challenges associated with development of mass spectrometry (MS)-based methods, as the corresponding mass shift is relatively small, +0.984 Da, and identical to the mass shift of deamidation. The focus of this review is to discuss recent advancements of citrullination-specific MS approaches and integration of the potential methodology for improved citrullination identification and characterization. In addition, the association of citrullination in disease networks is also highlighted.

Definitive Screening Designs to Optimize Library-Free DIA-MS Identification and Quantification of Neuropeptides.

Method optimization is crucial for successful mass spectrometry (MS) analysis. However, extensive method assessments, altering various parameters individually, are rarely performed due to practical limitations regarding time and sample quantity. To maximize sample space for optimization while maintaining reasonable instrumentation requirements, a definitive screening design (DSD) is leveraged for systematic optimization of data-independent acquisition (DIA) parameters to maximize crustacean neuropeptide identifications. While DSDs require several injections, a library-free methodology enables surrogate sample usage for comprehensive optimization of MS parameters to assess biomolecules from limited samples. We identified several parameters contributing significant first- or second-order effects to method performance, and the DSD model predicted ideal values to implement. These increased reproducibility and detection capabilities enabled the identification of 461 peptides, compared to 375 and 262 peptides identified through data-dependent acquisition (DDA) and a published DIA method for crustacean neuropeptides, respectively. Herein, we demonstrate a DSD optimization workflow, using standard material, not reliant on spectral libraries for the analysis of any low abundance molecules from previous samples of limited availability. This extends the DIA method to low abundance isoforms dysregulated or only detectable in disease samples, thus improving characterization of previously inaccessible biomolecules, such as neuropeptides. Data are available via ProteomeXchange with identifier PXD038520.

HyPep: An Open-Source Software for Identification and Discovery of Neuropeptides Using Sequence Homology Search.

Neuropeptides are a class of endogenous peptides that have key regulatory roles in biochemical, physiological, and behavioral processes. Mass spectrometry analyses of neuropeptides often rely on protein informatics tools for database searching and peptide identification. As neuropeptide databases are typically experimentally built and comprised of short sequences with high sequence similarity to each other, we developed a novel database searching tool, HyPep, which utilizes sequence homology searching for peptide identification. HyPep aligns de novo sequenced peptides, generated through PEAKS software, with neuropeptide database sequences and identifies neuropeptides based on the alignment score. HyPep performance was optimized using LC-MS/MS measurements of peptide extracts from various Callinectes sapidus neuronal tissue types and compared with a commercial database searching software, PEAKS DB. HyPep identified more neuropeptides from each tissue type than PEAKS DB at 1% false discovery rate, and the false match rate from both programs was 2%. In addition to identification, this report describes how HyPep can aid in the discovery of novel neuropeptides.

DiLeu Isobaric Labeling Coupled with Limited Proteolysis Mass Spectrometry for High-Throughput Profiling of Protein Structural Changes in Alzheimer's Disease.

High-throughput quantitative analysis of protein conformational changes has a profound impact on our understanding of the pathological mechanisms of Alzheimer's disease (AD). To establish an effective workflow enabling quantitative analysis of changes in protein conformation within multiple samples simultaneously, here we report the combination of N,N-dimethyl leucine (DiLeu) isobaric tag labeling with limited proteolysis mass spectrometry (DiLeu-LiP-MS) for high-throughput structural protein quantitation in serum samples collected from AD patients and control donors. Twenty-three proteins were discovered to undergo structural changes, mapping to 35 unique conformotypic peptides with significant changes between the AD group and the control group. Seven out of 23 proteins, including CO3, CO9, C4BPA, APOA1, APOA4, C1R, and APOA, exhibited a potential correlation with AD. Moreover, we found that complement proteins (e.g., CO3, CO9, and C4BPA) related to AD exhibited elevated levels in the AD group compared to those in the control group. These results provide evidence that the established DiLeu-LiP-MS method can be used for high-throughput structural protein quantitation, which also showed great potential in achieving large-scale and in-depth quantitative analysis of protein conformational changes in other biological systems.

Palliative Care Training for Geriatric Psychiatry Fellows: A National Survey Project.

OBJECTIVES: Palliative care is an essential part of the standard of care for individuals with serious medical illnesses. Integration of palliative care and mental health is important for elderly patients with medical and psychiatric comorbidities. Geriatric psychiatrists are natural stewards of palliative care-mental health integration, however this is contingent on palliative care training. Currently, palliative care training in geriatric psychiatry fellowship programs is uncharacterized. We surveyed geriatric psychiatry fellowship program directors in the United States to assess current palliative care training practices. METHODS: Web-based anonymous survey of geriatric psychiatry fellowship training directors RESULTS: Forty-six percent (28/61) of program directors responded. Seventy one percent (20/28) of programs provide didactics on palliative care. Seventy-seven percent (20/26) of programs provide clinical experiences in palliative care. Sixty-three percent (15/24) have formalized interactions between geriatric psychiatry and palliative care fellows. CONCLUSIONS: Palliative care training for geriatric psychiatry fellows is robust but unstandardized. Operationalizing palliative care training for geriatric psychiatrists may improve mental health integration into serious illness care.

Short chain α-pyrones capable of potentiating penicillin G against Pseudomonas aeruginosa.

The dramatic increase in bacterial resistance over the past three decades has greatly reduced the effectiveness of nearly all clinical antibiotics, bringing infectious disease to the forefront as a dire threat to global health. To combat these infections, adjuvant therapies have emerged as a way to reactivate known antibiotics against resistant pathogens. Herein, we report the evaluation of simplified α-pyrone adjuvants capable of potentiating penicillin G against Pseudomonas aeruginosa, a Gram-negative pathogen whose multidrug-resistant strains have been labeled by the Centers for Disease Control and Prevention as a serious threat to public health.

End-of-Life Care in Individuals With Serious Mental Illness.

BACKGROUND: Approximately 4.5% of the population live with serious mental illness (SMI), a term referring to mental health disorders that are chronic, impair function, and require ongoing treatment. People living with SMI are at risk of premature mortality relative to people without SMI. Chronic medical illnesses contribute significantly to mortality among individuals with SMI. The standard of care for individuals with serious medical illnesses includes palliative care. However, the provision of palliative care has not been operationalized for individuals with SMI. OBJECTIVES: To review existing data on end-of-life and palliative care for individuals with serious medical illness and comorbid serious mental illness. To operationalize the role of the consultation-liaison (C-L) psychiatrist in such care, with a particular eye towards redressing disparities. METHODS: In this narrative review, we draw upon a review of the literature on end-of-life and palliative care provision for individuals with serious medical illness and comorbid serious medical illness. We also draw upon the experiences of the authors in formulating best practices for the care of such patients. RESULTS: Individuals with SMI are at risk of suboptimal end-of-life care. Patient, clinician, and system-level factors all contribute to disparities including decreased access to palliative care, uneven continued engagement with mental health services, and low rates of advance care planning. C-L psychiatrists can use their expertise at the intersection of medicine and psychiatry to address such disparities by (1) correcting misassumptions, (2) promoting advance care planning, (3) engaging long-term caregivers, (4) recognizing social needs, (5) ensuring ongoing access to psychiatric treatment, and (6) addressing suffering. CONCLUSIONS: There are significant disparities in the end-of-life care of individuals with SMI. C-L psychiatrists have expertise to ally with medical providers and redress these disparities.

Bridge: Person-Centered Collaborative Care for Patients with Serious Mental Illness and Cancer.

BACKGROUND: Individuals with serious mental illness (SMI) experience increased cancer mortality due to inequities in cancer treatment. Psychiatric care at cancer diagnosis may improve care delivery, yet models for integrating psychiatry and cancer care are lacking. We assessed the feasibility and acceptability of a person-centered collaborative care trial for SMI and cancer. SUBJECTS, MATERIALS, AND METHODS: We developed the Bridge intervention for patients with SMI (schizophrenia, bipolar disorder, and severe major depression) and cancer. Bridge includes proactive identification of SMI, person-centered care from a psychiatrist and case manager, and collaboration with oncology. We conducted a 12-week, single-group trial in patients with SMI and a new breast, gastrointestinal, lung, or head/neck cancer. We assessed the feasibility of patient identification, enrollment and study completion; evaluated acceptability and perceived benefit with exit interviews with patients, caregivers, and oncology clinicians; and examined change in psychiatric symptoms with the Brief Psychiatric Rating Scale (BPRS). RESULTS: From November 2015 to April 2016, 30/33 eligible patients (90.9%) enrolled, and 25/29 (86.2%) completed assessments at all timepoints, meeting feasibility criteria. Of 24 patients, 23 (95.8%) found meeting with the psychiatrist helpful; 16/19 caregivers (84.2%) shared that Bridge addressed key caregiving challenges. Oncology clinicians evaluated Bridge as "very" or "most" useful for 94.3% of patients. Exit interviews with all participant groups suggested that Bridge fostered patient-clinician trust, increased access to psychiatric treatment, and enabled patients to initiate and complete cancer treatment. Psychiatric symptoms on the BPRS improved from baseline to 12 weeks. CONCLUSION: Bridge is a feasible and acceptable care delivery model for patients with SMI, their caregivers, and oncology clinicians. Randomized trials are warranted to assess the efficacy of improving cancer outcomes in this underserved population. IMPLICATIONS FOR PRACTICE: Serious mental illness affects 13 million U.S. adults who experience increased cancer mortality. To improve outcomes, new models of integrated oncology and mental health care are urgently needed. This study found that it was feasible to identify, enroll, and retain patients with serious mental illness and a new cancer in a trial of integrated mental health and cancer care (Bridge). Patients, caregivers, and oncologists reported that Bridge facilitated the initiation and completion of cancer care. Randomized trials are warranted to investigate the impact on cancer outcomes. Trial procedures may inform consent, engagement, and trial retention for patients with mental illness.

Development and evaluation of a six-day training program in supportive oncology research.

OBJECTIVE: Early career investigators have few opportunities for targeted training in supportive oncology research. To address this need, we developed, implemented, and evaluated an intensive, six-day workshop on methods in supportive oncology research for trainees and junior faculty across multiple disciplines. METHOD: A multidisciplinary team of supportive oncology researchers developed a workshop patterned after the clinical trials workshop offered jointly by the American Society of Clinical Oncology and American Association of Cancer Research. The curriculum included lectures and a mentored experience of writing a research protocol. Each year since 2015, the workshop has accepted and trained 36 early career investigators. Over the course of the workshop, participants present sections of their research protocols daily in small groups led by senior researchers, and have dedicated time to write and revise these sections. Primary outcomes for the workshop included the frequency of completed protocols by the end of the workshop, a pre- and posttest assessing participant knowledge, and follow-up surveys of the participants and their primary mentors.ResultOver three years, the workshop received 195 applications; 109 early career researchers were competitively selected to participate. All participants (109/109, 100%) completed writing a protocol by the end of their workshop. Participants and their primary mentors reported significant improvements in their research knowledge and skills. Each year, participants rated the workshop highly in terms of satisfaction, value, and likelihood of recommending it to a colleague. One year after the first workshop, most respondents (29/30, 96.7%) had either submitted their protocol or written at least one other protocol.Significance of resultsWe developed a workshop on research methods in supportive oncology. More early career investigators applied for the workshop than capacity, and the workshop was fully attended each year. Both the workshop participants and their primary mentors reported improvement in research skills and knowledge.

Predictors of Disruptions in Breast Cancer Care for Individuals with Schizophrenia.

BACKGROUND: Patients with schizophrenia experience markedly increased breast cancer mortality, yet reasons for this disparity are poorly understood. We sought to characterize disruptions in breast cancer care for patients with schizophrenia and identify modifiable predictors of those disruptions. MATERIALS AND METHODS: We performed a medical record review of 95 patients with schizophrenia and breast cancer treated at an academic cancer center between 1993 and 2015. We defined cancer care disruptions as processes that interfere with guideline-concordant cancer care, including delays to diagnosis or treatment, deviations from stage-appropriate treatment, and interruptions in treatment. We hypothesized that lack of psychiatric treatment at cancer diagnosis would be associated with care disruptions. RESULTS: Half of patients with schizophrenia experienced at least one breast cancer care disruption. Deviations in stage-appropriate treatment were associated with breast cancer recurrence at 5 years (p = .045). Patients without a documented psychiatrist experienced more delays (p = .016), without documented antipsychotic medication experienced more deviations (p = .007), and with psychiatric hospitalizations after cancer diagnosis experienced more interruptions (p < .0001). Independent of stage, age, and documented primary care physician, lack of documented antipsychotic medication (odds ratio [OR] = 4.97, 95% confidence interval [CI] = 1.90, 12.98) and psychiatric care (OR = 4.56, 95% CI = 1.37, 15.15) predicted cancer care disruptions. CONCLUSION: Disruptions in breast cancer care are common for patients with schizophrenia and are associated with adverse outcomes, including cancer recurrence. Access to psychiatric treatment at cancer diagnosis may protect against critical disruptions in cancer care for this underserved population. IMPLICATIONS FOR PRACTICE: Disruptions in breast cancer care are common for patients with schizophrenia, yet access to mental health treatment is rarely integrated into cancer care. When oncologists documented a treating psychiatrist and antipsychotic medication, patients had fewer disruptions in breast cancer care after adjusting for age, cancer stage, and access to primary care. Addressing psychiatric comorbidity at breast cancer diagnosis may increase the likelihood that patients with schizophrenia receive timely, stage-appropriate cancer treatment. Comanagement of schizophrenia and breast cancer at cancer diagnosis may be one key strategy to decrease inequities in cancer treatment and improve cancer survival in this underserved population.

Variation in blood serum antifreeze activity of Antarctic Trematomus fishes across habitat temperature and depth.

High latitude waters in the Southern Ocean can be near their freezing point and remain ice-covered throughout the year whereas lower latitude Southern Ocean waters have seasonal ice coverage and comparatively large (6 °C) annual temperature changes. The genus Trematomus (suborder Notothenioidei) is regarded primarily as a high latitude group because of its abundance there, they also inhabit the warmer regions in smaller numbers. Freeze avoidance in the notothenioids is linked to the presence of two antifreeze proteins (AFPs); the antifreeze glycoproteins (AFGPs) and antifreeze potentiating protein (AFPP), both of which adsorb to internal ice crystals inhibiting growth. Both high and low latitude trematomids possess sufficient AFP to lower their blood freezing point below that of seawater (-1.9 °C). We investigated the contributions of AFGPs and AFPP to the blood freezing point depression to determine how they varied with depth, water temperature, and the presence of ice. High latitude trematomids had lower blood freezing points than those inhabiting lower latitude waters indicating differences in their freeze avoidance capacities. Lower freezing points were associated with higher levels of antifreeze activity due to higher levels of both AFGP and AFPP. Populations of Trematomus hansoni and Trematomus bernacchii from shallow depths appear more freeze avoidant than populations inhabiting deep, ice-free water based on their lower freezing points and higher antifreeze activities. Gel electrophoresis of the trichloroacetic acid-soluble AFGPs indicates that only high molecular weight isoforms, which contribute more to AFGP activity, vary across species as well as between individuals of a species.

MotifQuest: An Automated Pipeline for Motif Database Creation to Improve Peptidomics Database Searching Programs.

Endogenous peptides are an abundant and versatile class of biomolecules with vital roles pertinent to the functionality of the nervous, endocrine, and immune systems and others. Mass spectrometry stands as a premier technique for identifying endogenous peptides, yet the field still faces challenges due to the lack of optimized computational resources for reliable raw mass spectra analysis and interpretation. Current database searching programs can exhibit discrepancies due to the unique properties of endogenous peptides, which typically require specialized search considerations. Herein, we present a high throughput, novel scoring algorithm for the extraction and ranking of conserved amino acid sequence motifs within any endogenous peptide database. Motifs are conserved patterns across organisms, representing sequence moieties crucial for biological functions, including maintenance of homeostasis. MotifQuest, our novel motif database generation algorithm, is designed to work in partnership with EndoGenius, a program optimized for database searching of endogenous peptides and that is powered by a motif database to capitalize on biological context to produce identifications. MotifQuest aims to quickly develop motif databases without any prior knowledge, a laborious task not possible with traditional sequence alignment resources. In this work we illustrate the utility of MotifQuest to expand EndoGenius' identification utility to other endogenous peptides by showcasing its ability to identify antimicrobial peptides. Additionally, we discuss the potential utility of MotifQuest to parse out motifs from a FASTA database file that can be further validated as new peptide drug candidates.

An Updated Guide to the Identification, Quantitation, and Imaging of the Crustacean Neuropeptidome.

Crustaceans serve as a useful, simplified model for studying peptides and neuromodulation, as they contain numerous neuropeptide homologs to mammals and enable electrophysiological studies at the single-cell and neural circuit levels. Crustaceans contain well-defined neural networks, including the stomatogastric ganglion, oesophageal ganglion, commissural ganglia, and several neuropeptide-rich organs such as the brain, pericardial organs, and sinus glands. As existing mass spectrometry (MS) methods are not readily amenable to neuropeptide studies, there is a great need for optimized sample preparation, data acquisition, and data analysis methods. Herein, we present a general workflow and detailed methods for MS-based neuropeptidomic analysis of crustacean tissue samples and circulating fluids. In conjunction with profiling, quantitation can also be performed with isotopic or isobaric labeling. Information regarding the localization patterns and changes of peptides can be studied via mass spectrometry imaging. Combining these sample preparation strategies and MS analytical techniques allows for a multi-faceted approach to obtaining deep knowledge of crustacean peptidergic signaling pathways.

Structural Proteomic Profiling of Cerebrospinal Fluids to Reveal Novel Conformational Biomarkers for Alzheimer's Disease.

Alzheimer's disease (AD) is the most common representation of dementia, with brain pathological hallmarks of protein abnormal aggregation, such as with amyloid beta and tau protein. It is well established that posttranslational modifications on tau protein, particularly phosphorylation, increase the likelihood of its aggregation and subsequent formation of neurofibrillary tangles, another hallmark of AD. As additional misfolded proteins presumably exist distinctly in AD disease states, which would serve as potential source of AD biomarkers, we used limited proteolysis-coupled with mass spectrometry (LiP-MS) to probe protein structural changes. After optimizing the LiP-MS conditions, we further applied this method to human cerebrospinal fluid specimens collected from healthy control, mild cognitive impairment (MCI), and AD subject groups to characterize proteome-wide misfolding tendencies as a result of disease progression. The fully tryptic peptides embedding LiP sites were compared with the half-tryptic peptides generated from internal cleavage of the same region to determine any structural unfolding or misfolding. We discovered hundreds of significantly up- and down-regulated peptides associated with MCI and AD indicating their potential structural changes in AD progression. Moreover, we detected 53 structurally changed regions in 12 proteins with high confidence between the healthy control and disease groups, illustrating the functional relevance of these proteins with AD progression. These newly discovered conformational biomarker candidates establish valuable future directions for exploring the molecular mechanism of designing therapeutic targets for AD.

Proteome-wide Profiling of Asymmetric Dimethylated Arginine in Human Breast Tumors.

Arginine methylation catalyzed by protein arginine methyltransferases (PRMTs) is a prevalent post-translational modification (PTM) that regulates diverse cellular processes. Aberrant expression of type I PRMTs that catalyze asymmetric arginine dimethylation (ADMA) is often found in cancer, though little is known about the ADMA status of substrate proteins in tumors. Using LC-MS/MS along with pan-specific ADMA antibodies, we performed global mapping of ADMA in five patient-derived xenograft (PDX) tumors representing different subtypes of human breast cancer. In total, 403 methylated sites from 213 proteins were identified, including 322 novel sites when compared to the PhosphositesPlus database. Moreover, using peptide arrays in vitro, approximately 70% of the putative substrates were validated to be methylated by PRMT1, PRMT4, and PRMT6. Notably, when compared with our previously identified ADMA sites from breast cancer cell lines, only 75 ADMA sites overlapped between cell lines and PDX tumors. Collectively, this study provides a useful resource for both PRMT and breast cancer communities for further exploitation of the functions of PRMT dysregulation during breast cancer progression.