Juneteenth in STEMM and the barriers to equitable science.

We are 52 Black scientists. Here, we establish the context of Juneteenth in STEMM and discuss the barriers Black scientists face, the struggles they endure, and the lack of recognition they receive. We review racism's history in science and provide institutional-level solutions to reduce the burdens on Black scientists.

A guide to establishing, implementing, and optimizing diversity, equity, inclusion, and accessibility (DEIA) committees.

Diversity, equity, inclusion, and accessibility (DEIA) efforts are increasingly recognized as critical for the success of academic institutions. These efforts are facilitated mainly through the formation of dedicated DEIA committees. DEIA committees enhance professional development and create a more inclusive environment, which benefits all members of the institution. Although leadership and faculty membership have recognized the importance and necessity of DEIA, the roles of DEIA committees may be more ambiguous. Although leadership and faculty may seek to support DEIA at their institutions, they may not always fully understand the necessity of these committees or how to successfully create a committee, foster and promote its success, and sustain its impact. Thus, here, we offer a background rationale and guide for strategically setting up DEIA committees for success and impact within an academic institution with applicability to scientific societies.

Exposing the Brain Proteomic Signatures of Alzheimer's Disease in Diverse Racial Groups: Leveraging Multiple Data Sets and Machine Learning.

Recent studies have highlighted that the proteome can be used to identify potential biomarker candidates for Alzheimer's disease (AD) in diverse cohorts. Furthermore, the racial and ethnic background of participants is an important factor to consider to ensure the effectiveness of potential biomarkers for representative populations. A promising approach to survey potential biomarker candidates for diagnosing AD in diverse cohorts is the application of machine learning to proteomics data sets. Herein, we leveraged six existing bottom-up proteomics data sets, which included non-Hispanic White, African American/Black, and Hispanic participants, to study protein changes in AD and cognitively unimpaired participants. Machine learning models were applied to these data sets and resulted in the identification of amyloid-ß precursor protein (APP) and heat shock protein ß-1 (HSPB1) as two proteins that have high ability to distinguish AD; however, each protein's performance varied based upon the racial and ethnic background of the participants. HSPB1 particularly was helpful for generating high areas under the curve (AUCs) for African American/Black participants. Overall, HSPB1 improved the performance of the machine learning models when combined with APP and/or participant age and is a potential candidate that should be further explored in AD biomarker discovery efforts.

Targeted Lipidomics To Measure Phospholipids and Sphingomyelins in Plasma: A Pilot Study To Understand the Impact of Race/Ethnicity in Alzheimer's Disease.

The number of people suffering from Alzheimer's disease (AD) is increasing rapidly every year. One aspect of AD that is often overlooked is the disproportionate incidence of AD among African American/Black populations. With the recent development of novel assays for lipidomics analysis in recent times, there has been a drastic increase in the number of studies focusing on changes of lipids in AD. However, very few of these studies have focused on or even included samples from African American/Black individuals samples. In this study, we aimed to determine if the lipidome in AD is universal across non-Hispanic White and African American/Black individuals. To accomplish this, a targeted mass spectrometry lipidomics analysis was performed on plasma samples (N = 113) obtained from cognitively normal (CN, N = 54) and AD (N = 59) individuals from African American/Black (N = 56) and non-Hispanic White (N = 57) backgrounds. Five lipids (PS 18:0_18:0, PS 18:0_20:0, PC 16:0_22:6, PC 18:0_22:6, and PS 18:1_22:6) were altered between AD and CN sample groups (p value < 0.05). Upon racial stratification, there were notable differences in lipids that were unique to African American/Black or non-Hispanic White individuals. PS 20:0_20:1 was reduced in AD in samples from non-Hispanic White but not African American/Black adults. We also tested whether race/ethnicity significantly modified the association between lipids and AD status by including a race × diagnosis interaction term in a linear regression model. PS 20:0_20:1 showed a significant interaction (p = 0.004). The discovery of lipid changes in AD in this study suggests that identifying relevant lipid biomarkers for diagnosis will require diversity in sample cohorts.

Brain expression of the vascular endothelial growth factor gene family in cognitive aging and alzheimer's disease.

Vascular endothelial growth factor (VEGF) is associated with the clinical manifestation of Alzheimer's disease (AD). However, the role of the VEGF gene family in neuroprotection is complex due to the number of biological pathways they regulate. This study explored associations between brain expression of VEGF genes with cognitive performance and AD pathology. Genetic, cognitive, and neuropathology data were acquired from the Religious Orders Study and Rush Memory and Aging Project. Expression of ten VEGF ligand and receptor genes was quantified using RNA sequencing of prefrontal cortex tissue. Global cognitive composite scores were calculated from 17 neuropsychological tests. ß-amyloid and tau burden were measured at autopsy. Participants (n = 531) included individuals with normal cognition (n = 180), mild cognitive impairment (n = 148), or AD dementia (n = 203). Mean age at death was 89 years and 37% were male. Higher prefrontal cortex expression of VEGFB, FLT4, FLT1, and PGF was associated with worse cognitive trajectories (p ≤ 0.01). Increased expression of VEGFB and FLT4 was also associated with lower cognition scores at the last visit before death (p ≤ 0.01). VEGFB, FLT4, and FLT1 were upregulated among AD dementia compared with normal cognition participants (p ≤ 0.03). All four genes associated with cognition related to elevated ß-amyloid (p ≤ 0.01) and/or tau burden (p ≤ 0.03). VEGF ligand and receptor genes, specifically genes relevant to FLT4 and FLT1 receptor signaling, are associated with cognition, longitudinal cognitive decline, and AD neuropathology. Future work should confirm these observations at the protein level to better understand how changes in VEGF transcription and translation relate to neurodegenerative disease.

Inclusion of African American/Black adults in a pilot brain proteomics study of Alzheimer's disease.

Alzheimer's disease (AD) disproportionately affects certain racial and ethnic subgroups, such as African American/Black and Hispanic adults. Genetic, comorbid, and socioeconomic risk factors contribute to this disparity; however, the molecular contributions have been largely unexplored. Herein, we conducted a pilot proteomics study of postmortem brains from African American/Black and non-Hispanic White adults neuropathologically diagnosed with AD compared to closely-matched cognitively normal individuals. Examination of hippocampus, inferior parietal lobule, and globus pallidus regions using quantitative proteomics resulted in 568 differentially-expressed proteins in AD. These proteins were consistent with the literature and included glial fibrillary acidic protein, peroxiredoxin-1, and annexin A5. In addition, 351 novel proteins in AD were identified, which could partially be due to cohort diversity. From linear regression analyses, we identified 185 proteins with significant race x diagnosis interactions across various brain regions. These differences generally were reflective of differential expression of proteins in AD that occurred in only a single racial/ethnic group. Overall, this pilot study suggests that disease understanding can be furthered by including diversity in racial/ethnic groups; however, this must be done on a larger scale.

Evaluating Combined Precursor Isotopic Labeling and Isobaric Tagging Performance on Orbitraps To Study the Peripheral Proteome of Alzheimer's Disease.

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is an enhanced multiplexing strategy currently capable of analyzing up to 24 samples simultaneously. This capability is especially helpful when studying multiple tissues and biological replicates in models of disease, such as Alzheimer's disease (AD). Here, cPILOT was used to study proteomes from heart, liver, and brain tissues in a late-stage amyloid precursor protein/presenilin-1 (APP/PS-1) human transgenic double-knock-in mouse model of AD. The original global cPILOT assay developed on an Orbitrap Velos instrument was transitioned to an Orbitrap Fusion Lumos instrument. The advantages of faster scan rates, lower limits of detection, and synchronous precursor selection on the Fusion Lumos afford greater numbers of isobarically tagged peptides to be quantified in comparison to the Orbitrap Velos. Parameters such as LC gradient, m/z isolation window, dynamic exclusion, targeted mass analyses, and synchronous precursor scan were optimized leading to >600 000 PSMs, corresponding to 6074 proteins. Overall, these studies inform of system-wide changes in brain, heart, and liver proteins from a mouse model of AD.

In Vivo Fast Photochemical Oxidation of Proteins Using Enhanced Multiplexing Proteomics.

In vivo fast photochemical oxidation of proteins (IV-FPOP) is a hydroxyl radical protein footprinting method used to study protein structure and protein-protein interactions. Oxidatively modified proteins by IV-FPOP are analyzed by mass spectrometry (MS), and the extent of oxidation is quantified by label-free MS. Peptide oxidation changes yield useful information about protein structure, due to changes in solvent accessibility. However, the sample size necessary for animal studies requires increased sample preparation and instrument time. Here, we report the combined application of IV-FPOP and the enhanced multiplexing strategy combined precursor isotopic labeling and isobaric tagging (cPILOT) for higher-throughput analysis of oxidative modifications in C. elegans. Key differences in the performance of label-free MS and cPILOT were identified. The addition of oxygen (+16) was the most abundant modification identified among all known possible FPOP modifications. This study presents IV-FPOP coupled with enhanced multiplexing strategies such as cPILOT to increase throughput of studies seeking to examine oxidative protein modifications.

Evaluating a targeted multiple reaction monitoring approach to global untargeted lipidomic analyses of human plasma.

RATIONALE: The Lipidyzer platform was recently updated on a SCIEX QTRAP 6500+ mass spectrometer and offers a targeted lipidomics assay including 1150 different lipids. We evaluated this targeted approach using human plasma samples and compared the results against a global untargeted lipidomics method using a high-resolution Q Exactive HF Orbitrap mass spectrometer. METHODS: Lipids from human plasma samples (N = 5) were extracted using a modified Bligh-Dyer approach. A global untargeted analysis was performed using a Thermo Orbitrap Q Exactive HF mass spectrometer, followed by data analysis using Progenesis QI software. Multiple reaction monitoring (MRM)-based targeted analysis was performed using a QTRAP 6500+ mass spectrometer, followed by data analysis using SCIEX OS software. The samples were injected on three separate days to assess reproducibility for both approaches. RESULTS: Overall, 465 lipids were identified from 11 lipid classes in both approaches, of which 159 were similar between the methods, 168 lipids were unique to the MRM approach, and 138 lipids were unique to the untargeted approach. Phosphatidylcholine and phosphatidylethanolamine species were the most commonly identified using the untargeted approach, while triacylglycerol species were the most commonly identified using the targeted MRM approach. The targeted MRM approach had more consistent relative abundances across the three days than the untargeted approach. Overall, the coefficient of variation for inter-day comparisons across all lipid classes was ∼ 23% for the untargeted approach and ∼ 9% for the targeted MRM approach. CONCLUSIONS: The targeted MRM approach identified similar numbers of lipids to a conventional untargeted approach, but had better representation of 11 lipid classes commonly identified by both approaches. Based on the separation methods employed, the conventional untargeted approach could better detect phosphatidylcholine and sphingomyelin lipid classes. The targeted MRM approach had lower inter-day variability than the untargeted approach when tested using a small group of plasma samples. These studies highlight the advantages in using targeted MRM approaches for human plasma lipidomics analysis.

A Diverse View of Science to Catalyse Change.

Valuing diversity leads to scientific excellence, the progress of science and most importantly, it is simply the right thing to do. We can value diversity not only in words, but also in actions.

The Potential of 'Omics to Link Lipid Metabolism and Genetic and Comorbidity Risk Factors of Alzheimer's Disease in African Americans.

Alzheimer's disease (AD) disproportionately affects African Americans (AAs) and Hispanics, who are more likely to have AD than non-Hispanic Whites (NHWs) and Asian Americans. Racial disparities in AD are multifactorial, with potential contributing factors including genetics, comorbidities, diet and lifestyle, education, healthcare access, and socioeconomic status. Interestingly, comorbidities such as hypertension, type 2 diabetes mellitus, and cardiovascular disease also impact AAs. It is plausible that a common underlying molecular basis to these higher incidences of AD and comorbidities exists especially among AAs. A likely common molecular pathway that is centrally linked to AD and these noted comorbidities is alterations in lipid metabolism. Several genes associated with AD risk-most notably, the ε4 allele of the apolipoprotein E (APOE) gene and several mutations in the ATP-binding cassette transporter A7 (ABCA7) gene-are linked to altered lipid metabolism, especially in AAs. This review explores the role of lipid metabolism in AD broadly, as well as in other comorbidities that are prevalent in AAs. Because there are gaps in our understanding of the molecular basis of higher incidences of AD in AAs, 'omics approaches such as proteomics and lipidomics are presented as potential methods to improve our knowledge in these areas.

Increased N,N-Dimethyl Leucine Isobaric Tag Multiplexing by a Combined Precursor Isotopic Labeling and Isobaric Tagging Approach.

Multiplex isobaric tags have become valuable tools for high-throughput quantitative analysis of complex biological samples in discovery-based proteomics studies. Hybrid labeling strategies that pair stable isotope mass difference labeling with multiplex isobaric tag-based quantification further facilitate these studies by greatly increasing multiplexing capability. In this work, we present a cost-effective chemical labeling approach that couples duplex stable isotope dimethyl labeling with our custom 12-plex N,N-dimethyl leucine (DiLeu) isobaric tags in a combined precursor isotopic labeling and isobaric tagging (cPILOT) strategy that is compatible with a wide variety of biological samples and permits 24-plex quantification in a single LC-MS/MS experiment. We demonstrate the utility of the DiLeu cPILOT approach by labeling yeast digests and performing proof-of-principle quantification experiments on the Orbitrap Fusion Lumos.

Multiplexing Biomarker Methods, Proteomics and Considerations for Alzheimer's Disease.

Biomarker research for Alzheimer's disease (AD) has been growing rapidly over recent years especially as the number of persons affected by this disease is nearing approximately 46 million worldwide. Single biomarker assays are challenging to establish since AD is multifactorial and complex. In addition to the classic signs of diminished cognition and memory, AD patients can also exhibit symptoms which may be confused with some psychiatric disorders, such as depression. No molecular biomarkers have been established or translated into clinical tools although recent efforts have resulted in addition of molecular biomarker profiles to the National Institute of Neurological and Communicative Disorders and Stroke and Alzheimer's Disease and Related Disorders Association criteria for research purposes. The three accepted molecular biomarkers are amyloid-ßeta peptide 1-42, total tau protein and hyperphosphorylated tau at threonine 181 in human cerebrospinal fluid (CSF). Aside from these three CSF markers, a number of potential candidates have been identified in CSF and other body fluids. In order to identify biomarkers for diagnosis, early prevention, prognosis and response to therapeutic treatment, multiplex biomarker tests will be required. These include multiplex immunoassay and mass spectrometry-based proteomics platforms. Proteomics analyses of bodily fluids such as plasma are growing in number and providing potential targets for further investigation and validation in AD research. This chapter highlights proteomic biomarker assays and their applications and potential use for clinical diagnosis and prognosis of AD.

High-throughput endogenous measurement of S-nitrosylation in Alzheimer's disease using oxidized cysteine-selective cPILOT.

Reversible cysteine modifications play important physiological roles such as modulating enzymatic catalysis, maintaining redox homeostasis and conducting cellular signaling. These roles can be critical in the context of disease. Oxidative modifications such as S-nitrosylation (SNO) are signatures of neurodestruction in conditions of oxidative stress however are also indicators of neuroprotection and normal signaling in cellular environments with low concentrations of reactive oxygen and nitrogen species. SNO is a dynamic and low abundance modification and requires sensitive and selective analytical methods for its detection in biological tissues. Here we present an enhanced multiplexing strategy to study SNO in complex mixtures arising from tissues. This method, termed oxidized cysteine-selective cPILOT (OxcyscPILOT), allows simultaneous analysis of SNO-modified peptides in 12 samples. OxcyscPILOT has three primary steps: (1) blocking of free thiols by a cysteine-reactive reagent, (2) enrichment of peptides containing SNO on a solid phase resin, and (3) isotopic labeling and isobaric tagging of enriched peptides on the solid phase resin. This approach offers the advantage of allowing total protein abundance levels to be measured simultaneously with endogenous SNO levels and measurement of SNO levels across four biological replicates in a single analysis. Furthermore, the relative amount of SNO on a specific cysteine site can also be determined. A well-known model of Alzheimer's disease, the APP/PS-1 transgenic mouse model, was selected for demonstration of the method as several SNO-modified proteins have previously been reported in brain and synaptosomes from AD subjects. OxcyscPILOT analysis resulted in identification of 138 SNO-modified cysteines in brain homogenates that correspond to 135 proteins. Many of these SNO-modified proteins were only present in wild-type or AD mice, whereas 93 proteins had SNO signals in both WT and AD. Pathway analysis links SNO-modified proteins to various biological pathways especially metabolism and signal transduction, consistent with previous reports in the literature. The OxcyscPILOT strategy provides enhanced multiplexing capability to current redox proteomics methods to study oxidative modifications of cysteine.

A simple isotopic labeling method to study cysteine oxidation in Alzheimer's disease: oxidized cysteine-selective dimethylation (OxcysDML).

Cysteine is widely involved in redox signaling pathways through a number of reversible and irreversible modifications. Reversible modifications (e.g., S-glutathionylation, S-nitrosylation, disulfide bonds, and sulfenic acid) are used to protect proteins from oxidative attack and maintain cellular homeostasis, while irreversible oxidations (e.g., sulfinic acid and sulfonic acid) serve as hallmarks of oxidative stress. Proteomic analysis of cysteine-enriched peptides coupled with reduction of oxidized thiols can be used to measure the oxidation states of cysteine, which is helpful for elucidating the role that oxidative stress plays in biology and disease. As an extension of our previously reported cysDML method, we have developed oxidized cysteine-selective dimethylation (OxcysDML), to investigate the site-specific total oxidation of cysteine residues in biologically relevant samples. OxcysDML employs (1) blocking of free thiols by a cysteine-reactive reagent, (2) enrichment of peptides containing reversibly oxidized cysteine by a solid phase resin, and (3) isotopic labeling of peptide amino groups to quantify cysteine modifications arising from different biological conditions. On-resin enrichment and labeling minimizes sample handing time and improves efficiency in comparison with other redox proteomic methods. OxcysDML is also inexpensive and flexible, as it can accommodate the exploration of various cysteine modifications. Here, we applied the method to liver tissues from a late-stage Alzheimer's disease (AD) mouse model and wild-type (WT) controls. Because we have previously characterized this proteome using the cysDML approach, we are able here to probe deeper into the redox status of cysteine in AD. OxcysDML identified 1129 cysteine sites (from 527 proteins), among which 828 cysteine sites underwent oxidative modifications. Nineteen oxidized cysteine sites had significant alteration levels in AD and represent proteins involved in metabolic processes. Overall, we have demonstrated OxcysDML as a simple, rapid, robust, and inexpensive redox proteomic approach that is useful for gaining deeper insight into the proteome of AD.

Mass spectrometry and redox proteomics: applications in disease.

Proteomics techniques are continuously being developed to further understanding of biology and disease. Many of the pathways that are relevant to disease mechanisms rely on the identification of post-translational modifications (PTMs) such as phosphorylation, acetylation, and glycosylation. Much attention has also been focused on oxidative PTMs which include protein carbonyls, protein nitration, and the incorporation of fatty acids and advanced glycation products to amino acid side chains, amongst others. The introduction of these PTMs in the cell can occur due to the attack of reactive oxygen and nitrogen species (ROS and RNS, respectively) on proteins. ROS and RNS can be present as a result of normal metabolic processes as well as external factors such as UV radiation, disease, and environmental toxins. The imbalance of ROS and RNS with antioxidant cellular defenses leads to a state of oxidative stress, which has been implicated in many diseases. Redox proteomics techniques have been used to characterize oxidative PTMs that result as a part of normal cell signaling processes as well as oxidative stress conditions. This review highlights many of the redox proteomics techniques which are currently available for several oxidative PTMs and brings to the reader's attention the application of redox proteomics for understanding disease pathogenesis in neurodegenerative disorders and others such as cancer, kidney, and heart diseases.

MS(3)-based quantitative proteomics using pulsed-Q dissociation.

RATIONALE: Isobaric tagging reagents, such as tandem mass tags (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ), are high-throughput methods that allow the analysis of multiple samples simultaneously, which reduces instrument time and error. Accuracy and precision of isobaric tags are limited, however, in tandem mass spectrometry (MS/MS) acquisition due to co-isolation and co-fragmentation of neighboring peptide peaks in precursor scans. Here we present a MS(3) method using pulsed-Q dissociation (PQD) in ion trap and Orbitrap instrumentation as a means to improve ratio distortion and maintain high numbers of identified and quantified proteins. METHODS: Mouse brain protein digests were labeled with TMT-128, 129, 130, 131 reagents, mixed in the following molar ratios 1:1:2:5, respectively, and analyzed using HCD-MS(3) and PQD-MS(3) methods. The most intense fragment ion (termed as HCD-MS(3)-top ion or PQD-MS(3)-top ion) or y1 ion (i.e., lysine-TMT tag ion; termed as HCD-MS(3)-y1 or PQD-MS(3)-y1) in collision-induced dissociation (CID) MS/MS was selected for MS(3). RESULTS: Calculated protein ratios obtained in HCD-MS(3)-top ion and PQD-MS(3)-top ion, HCD-MS(3)-y1, and PQD-MS(3)-y1 are accurate and PQD-MS(3) methods resulted in higher numbers of identified and quantified peptide spectral counts and proteins. CONCLUSIONS: PQD-MS(3) methods increase the amount of MS/MS spectra collected and number of quantified proteins and are accessible to those researchers with not only an orbitrap but also an ion trap mass spectrometer.

Proteome characterization of splenocytes from an Aßpp/ps-1 Alzheimer's disease model.

Alzheimer's disease (AD) is the sixth leading cause of US deaths. In addition to neurodegenerative deficits in AD, changes in the immune system have also been observed. Proteomic analysis of specific immune cell populations may help gain insights into mechanisms of peripheral immunity in AD. Herein, we report proteome characterization for two subsets of splenocytes (i.e. CD90+ cells and a heterogeneous pool of CD90- cells) from a double transgenic mutant amyloid precursor protein/presenilin-1 (Aßpp/ps-1) AD mouse model. Overall, 906 proteins were identified from both cell types with 275 and 334 proteins uniquely identified as CD90+ and CD90- cells, respectively. Proteins identified in CD90+ and CD90- cells were significantly involved in 18 and 19 KEGG pathways, respectively. Amongst these, pathways associated with AD and antigen processing and presentation were identified in CD90+ and CD90- subsets, respectively. This is the first study to provide a reference proteome map for splenocyte populations in Aßpp/ps-1 double transgenic mice which will be helpful for future studies focused on understanding peripheral changes in this model. All MS data have been deposited in the ProteomeXchange with identifier PXD000203 (http://proteomecentral.proteomexchange.org/dataset/PXD000203).

Proteomics reveals age-related differences in the host immune response to sepsis.

Sepsis is commonly caused by community-acquired pneumonia (CAP) and may develop into severe sepsis, characterized by multiple organ failure. The risk of severe sepsis among CAP patients and subsequent mortality increases sharply after the age of 65. The molecular mechanisms associated with this age-related risk are not fully understood. To better understand factors involved with increased incidence and mortality of severe sepsis in the elderly, we used a nested case-control study of patients enrolled in a multicenter observational cohort of 2320 participants with CAP. We identified a total of 39 CAP patients 50-65 and 70-85 years old who did or did not develop severe sepsis. Plasma samples were obtained on presentation to the emergency department and prior to therapeutic interventions. A semiquantitative plasma proteomics workflow was applied which incorporated tandem immunoaffinity depletion, iTRAQ labeling, strong cation exchange fractionation, and nanoflow liquid chromatography coupled to high-resolution mass spectrometry. In total, 772 proteins were identified, of which 58 proteins exhibit statistically significant differences in expression levels among patients with severe sepsis as a function of age. Differentially expressed proteins are involved in pathways such as acute phase response, coagulation signaling, atherosclerosis signaling, lipid metabolism, and production of nitric oxide and reactive oxygen species. This study provides insight into factors that may explain age-related differences in incidence of severe sepsis in the elderly.