Biological mass spectrometry enables spatiotemporal 'omics: From tissues to cells to organelles.

Biological processes unfold across broad spatial and temporal dimensions, and measurement of the underlying molecular world is essential to their understanding. Interdisciplinary efforts advanced mass spectrometry (MS) into a tour de force for assessing virtually all levels of the molecular architecture, some in exquisite detection sensitivity and scalability in space-time. In this review, we offer vignettes of milestones in technology innovations that ushered sample collection and processing, chemical separation, ionization, and 'omics analyses to progressively finer resolutions in the realms of tissue biopsies and limited cell populations, single cells, and subcellular organelles. Also highlighted are methodologies that empowered the acquisition and analysis of multidimensional MS data sets to reveal proteomes, peptidomes, and metabolomes in ever-deepening coverage in these limited and dynamic specimens. In pursuit of richer knowledge of biological processes, we discuss efforts pioneering the integration of orthogonal approaches from molecular and functional studies, both within and beyond MS. With established and emerging community-wide efforts ensuring scientific rigor and reproducibility, spatiotemporal MS emerged as an exciting and powerful resource to study biological systems in space-time.

Activity Guided Azide-methyllysine Photo-trapping for Substrate Profiling of Lysine Demethylases.

Reversible post-translational modifications (PTMs) are key to establishing protein-protein and protein-nucleic acid interactions that govern a majority of the signaling pathways in cells. Sequence-specific PTMs are catalyzed by transferases, and their removal is carried out by a class of reverse-acting enzymes termed "detransferases". Currently available chemoproteomic approaches have been valuable in characterizing substrates of transferases. However, proteome-wide cataloging of the substrates of detransferases is challenging, mostly due to the loss of the epitope, rendering immunoprecipitation and activity-based methods ineffective. Herein, we develop a general chemoproteomic strategy called crosslinking-assisted substrate identification (CASI) for systematic characterization of cellular targets of detransferases and successfully apply it to lysine demethylases (KDMs) which catalyze the removal of methyl groups from lysine sidechain in histones to modulate gene transcription. By setting up a targeted azido-methylamino photo-reaction deep inside the active site of KDM4, engineered to carry p-azido phenylalanine, we reveal a novel "demethylome" that has escaped the traditional methods. The proteomic survey led to the identification of a battery of nonhistone substrates of KDM4, extending the biological footprint of KDM4 beyond its canonical functions in gene transcription. A notable finding of KDM4A-mediated demethylation of an evolutionarily conserved lysine residue in eukaryotic translational initiation factor argues for a much broader role of KDM4A in ribosomal processes. CASI, representing a substantive departure from earlier approaches by shifting focus from simple peptide-based probes to employing full-length photo-activatable demethylases, is poised to be applied to >400 human detransferases, many of which have remained poorly understood due to the lack of knowledge about their cellular targets.

Mapping protein-exopolysaccharide binding interaction in Staphylococcus epidermidis biofilms by live cell proximity labeling.

Bacterial biofilms consist of cells encased in an extracellular polymeric substance (EPS) composed of exopolysaccharides, extracellular DNA, and proteins that are critical for cell-cell adhesion and protect the cells from environmental stress, antibiotic treatments, and the host immune response. Degrading EPS components or blocking their production have emerged as promising strategies for prevention or dispersal of bacterial biofilms, but we still have little information about the specific biomolecular interactions that occur between cells and EPS components and how those interactions contribute to biofilm production. Staphylococcus epidermidis is a leading cause of nosocomial infections as a result of producing biofilms that use the exopolysaccharide poly-(1→6)-ß-N-acetylglucosamine (PNAG) as a major structural component. In this study, we have developed a live cell proximity labeling approach combined with quantitative mass spectrometry-based proteomics to map the PNAG interactome of live S. epidermidis biofilms. Through these measurements we discovered elastin-binding protein (EbpS) as a major PNAG-interacting protein. Using live cell binding measurements, we found that the lysin motif (LysM) domain of EbpS specifically binds to PNAG present in S. epidermidis biofilms. Our work provides a novel method for the rapid identification of exopolysaccharide-binding proteins in live biofilms that will help to extend our understanding of the biomolecular interactions that are required for bacterial biofilm formation.

Deep Proteome of the Developing Chick Midbrain.

The epithelial-to-mesenchymal transition (EMT) and migration of cranial neural crest cells within the midbrain are critical processes that permit proper craniofacial patterning in the early embryo. Disruptions in these processes not only impair development but also lead to various diseases, underscoring the need for their detailed understanding at the molecular level. The chick embryo has served historically as an excellent model for human embryonic development, including cranial neural crest cell EMT and migration. While these developmental events have been characterized transcriptionally, studies at the protein level have not been undertaken to date. Here, we applied mass spectrometry (MS)-based proteomics to establish a deep proteomics profile of the chick midbrain region during early embryonic development. Our proteomics method combines optimal lysis conditions, offline fractionation, separation on a nanopatterned stationary phase (µPAC) using nanoflow liquid chromatography, and detection using quadrupole-ion trap-Orbitrap tribrid high-resolution tandem MS. Identification of >5900 proteins and >450 phosphoproteins in this study marks the deepest coverage of the chick midbrain proteome to date. These proteins have known roles in pathways related to neural crest cell EMT and migration such as signaling, proteolysis/extracellular matrix remodeling, and transcriptional regulation. This study offers valuable insight into important developmental processes occurring in the midbrain region and demonstrates the utility of proteomics for characterization of tissue microenvironments during chick embryogenesis.

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.

ABCA7, a Genetic Risk Factor Associated with Alzheimer's Disease Risk in African Americans.

African American/Black adults are twice as likely to have Alzheimer's disease (AD) compared to non-Hispanic White adults. Genetics partially contributes to this disparity in AD risk, among other factors, as there are several genetic variants associated with AD that are more prevalent in individuals of African or European ancestry. The phospholipid-transporting ATPase ABCA7 (ABCA7) gene has stronger associations with AD risk in individuals with African ancestry than in individuals with European ancestry. In fact, ABCA7 has been shown to have a stronger effect size than the apolipoprotein E (APOE) ɛ4 allele in African American/Black adults. ABCA7 is a transmembrane protein involved in lipid homeostasis and phagocytosis. ABCA7 dysfunction is associated with increased amyloid-beta production, reduced amyloid-beta clearance, impaired microglial response to inflammation, and endoplasmic reticulum stress. This review explores the impact of ABCA7 mutations that increase AD risk in African American/Black adults on ABCA7 structure and function and their contributions to AD pathogenesis. The combination of biochemical/biophysical and 'omics-based studies of these variants needed to elucidate their downstream impact and molecular contributions to AD pathogenesis is highlighted.

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.

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.