Biology subjects

Westphall, M. S.

Publications and source records attributed to Westphall, M. S..

8 recordsLinked to original sources

bioRxiv2024

Boosting the Sensitivity of Quantitative Single-Cell Proteomics with Activated Ion-Tandem Mass Tags (AI-TMT)

Single-cell proteomics is a powerful approach to precisely profile protein landscapes within individual cells toward a comprehensive understanding of proteomic functions and tissue and cellular states. The inherent challenges associated with limited starting material in single-cell analyses demands heightened analytical sensitivity. Just as advances in sample preparation maximize the amount of material that makes it from the cell to the mass spectrometer, we strive to maximize the number of ions that make it from ion source to the detector. In isobaric tagging experiments, limited reporter ion generation limits quantitative accuracy and precision. The combination of infrared photoactivation and ion parking circumvents the m/z dependence inherent in HCD, maximizing reporter generation and avoiding unintended degradation of TMT reporter molecules in a method we term activated ion-tandem mass tags (AI-TMT). The method was applied to single-cell human proteomes using 18-plex TMTpro, resulting in a 4-5-fold increase in reporter ion signal on average compared to conventional SPS-MS3 approaches. AI-TMT enables faster duty cycles, higher throughput, and increased peptide identification and quantification. Comparative experiments showcase 4-5-fold lower injection times for AI-TMT, providing superior sensitivity without compromising accuracy. In all, AI-TMT enhances the sensitivity and dynamic range of proteomic experiments and is compatible with other techniques, including gas-phase fractionation and real-time searching, promising increased gains in the study of cellular heterogeneity and disease mechanisms.

biochemistry↗

bioRxiv2023

One-Hour Phosphoproteome Analysis with the Orbitrap Astral Mass Spectrometer

Owing to its roles in cellular signal transduction, protein phosphorylation plays critical roles in myriad cell processes. That said, detecting and quantifying protein phosphorylation has remained a challenge. We describe the use of a novel mass spectrometer (Orbitrap Astral) coupled with data-independent acquisition (DIA) to achieve rapid and deep analysis of human and mouse phosphoproteomes. With this method we map approximately 30,000 unique human phosphorylation sites within a half-hour of data collection. The technology was benchmarked to other state-of-the-art MS platforms using both synthetic peptide standards and with EGF-stimulated HeLa cells. We applied this approach to generate a phosphoproteome multi-tissue atlas of the mouse. Altogether, we detected 81,120 unique phosphorylation sites within 12 hours of measurement. With this unique dataset, we examine the sequence, structural, and kinase specificity context of protein phosphorylation. Finally, we highlight the discovery potential of this resource with multiple examples of novel phosphorylation events relevant to mitochondrial and brain biology.

systems biology↗

bioRxiv2023

Cryogenic Soft Landing Improves Structural Preservation of Protein Complexes

We describe an apparatus for the cryogenic landing of particles from the ion beam of a mass spectrometer onto transmission electron microscope grids for cryo-electron microscopy. This system also allows for the controlled formation of thin films of amorphous ice on the grid surface. We demonstrate that as compared to room temperature landings, use of this cryogenic landing device greatly improves the structural preservation of deposited protein-protein complexes. Further, landing under cryogenic conditions can increase the diversity of particle orientations, allowing for improved 3D structural interpretation. Finally, we conclude that this approach allows for the direct coupling of mass spectrometry with cryo-electron microscopy.

biochemistry↗

bioRxiv2021

3D Structure Determination of Protein Complexes using Matrix-Landing Mass Spectrometry

Native mass spectrometry (MS) is an emerging technology that can provide complementary data to electron microscopy (EM) for protein structure characterization. Beyond the ability to provide mass measurements of gas-phase biomolecular ions, MS instruments offer the ability to purify, select, and precisely control the spatial location of these ions. Here we present a modified Orbitrap MS system capable of depositing a native MS ion beam onto EM grids. We further describe use of a chemical landing matrix that both preserves and protects the structural integrity of the deposited particles. With this system we obtained the first 3D reconstructed structure of gas-phase, deposited biomolecular ions - the 800 KDa protein complex GroEL. These data provide direct evidence that non-covalent protein complexes can indeed retain their condensed-phase structures following ionization and vaporization. Finally, we describe how further developments of this technology could pave the way to an integrated MS-EM technology with promise to provide improved cryo-EM sample preparation over conventional plunge-freezing techniques.

molecular biology↗

bioRxiv2020

Proteomics, lipidomics, metabolomics and 16S rDNA sequencing of dental plaque from patients with diabetes and periodontal disease

Oral microbiome influences human health, specifically pre- and type 2 diabetes (Pre-DM/DM) and periodontal diseases (PD), through complex microbial interactions. To explore these relations, we performed 16S rDNA sequencing, metabolomics, lipidomics, and proteomics analyses on supragingival dental plaque collected from individuals with Pre-DM/DM (n=39), Pre-DM/DM and PD (n=37), PD alone (n=11), or neither (n=10). We identified on average 2,790 operational taxonomic units and 2,025 microbial and host proteins per sample and quantified 110 metabolites and 415 lipids. Plaque samples from Pre-DM/DM patients contained higher abundance of Fusobacterium and Tannerella vs. plaques from metabolically healthy. Phosphatidylcholines, plasmenyl-phosphatidylcholines, ceramides containing non-OH fatty acids, and host proteins related to actin filament rearrangement were elevated in plaques from PD vs. non-PD. Cross-omic correlation analysis enabled the detection of a strong association between Lautropia and mono-methyl phophospotidlyethanolamine (PE-NMe), striking because synthesis of PE-NMe is uncommon in oral bacteria. Lipidomics analysis of in vitro cultures of Lautropia mirabilis confirmed the bacterias synthesis of PE-NMe. This comprehensive analysis revealed a novel microbial metabolic pathway and significant associations of host-derived proteins with PD.

systems biology↗

bioRxiv2020

Mapping physiological ADP-ribosylation using Activated Ion Electron Transfer Dissociation (AI-ETD)

ADP-ribosylation (ADPr) is a post-translational modification that plays pivotal roles in a wide range of cellular processes. Mass spectrometry (MS)-based analysis of ADPr under physiological conditions, without relying on genetic or chemical perturbation, has been hindered by technical limitations. Here, we describe the applicability of Activated Ion Electron Transfer Dissociation (AI-ETD) for MS-based proteomics analysis of physiological ADPr using our unbiased Af1521 enrichment strategy. To benchmark AI-ETD, we profiled 9,000 ADPr peptides mapping to >5,000 unique ADPr sites from a limited number of cells exposed to oxidative stress, corresponding to 120% and 28% more ADPr peptides compared to contemporary strategies using ETD and EThcD, respectively. Under physiological conditions AI-ETD identified 450 ADPr sites on low-abundant proteins, including in vivo cysteine auto-modifications on PARP8 and tyrosine auto-modifications on PARP14, hinting at specialist enzymatic functions for these enzymes. Collectively, our data provides new insights into the physiological regulation of ADP-ribosylation.

molecular biology↗

bioRxiv2019

Real time health monitoring through urine metabolomics

Current healthcare practices are reactive and based on limited physiological information collected months or years apart. By enabling patients and healthy consumers access to continuous measurements of health, wearable devices and digital medicine stand to realize highly personalized and preventative care. However, most current digital technologies provide information on a limited set of physiological traits, such as heart rate and step count, which alone offer little insight into the etiology of most diseases. Here we propose to integrate data from biohealth smartphone applications with continuous metabolic phenotypes derived from urine metabolites. This combination of molecular phenotypes with quantitative measurements of lifestyle reflect the biological consequences of human behavior in real time. We present data from an observational study involving two healthy subjects and discuss the challenges, opportunities, and implications of integrating this new layer of physiological information into digital medicine. Though our dataset is limited to two subjects, our analysis (also available through an interactive web-based visualization tool) provides an initial framework to monitor lifestyle factors, such as nutrition, drug metabolism, exercise, and sleep using urine metabolites.

biochemistry↗

bioRxiv2019

Capturing site-specific heterogeneity with large-scale N-glycoproteome analysis

Protein glycosylation is a highly important, yet a poorly understood protein post-translational modification. Thousands of possible glycan structures and compositions create potential for tremendous site heterogeneity and analytical challenge. A lack of suitable analytical methods for large-scale analyses of intact glycopeptides has ultimately limited our abilities to both address the degree of heterogeneity across the glycoproteome and to understand how it contributes biologically to complex systems. Here we show that N-glycoproteome site-specific microheterogeneity can be captured via large-scale glycopeptide profiling with methods enabled by activated ion electron transfer dissociation (AI-ETD), ultimately characterizing 1,545 N-glycosites (>5,600 unique N-glycopeptides) from mouse brain tissue. Moreover, we have used this large-scale glycoproteomic data to develop several new visualizations that will prove useful for analyzing intact glycopeptides in future studies. Our data reveal that N-glycosylation profiles can differ between subcellular regions and structural domains and that N-glycosite heterogeneity manifests in several different forms, including dramatic differences in glycosites on the same protein.

systems biology↗