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23rd Human Proteome Organization World Congress

Top-down proteomics of 10,000 single brain cells

Professor

Keynote speaker

Keynote lecture

Top-down characterization of genetically encoded Bcl-XL phosphorylation and phosphomimetics using electron capture dissociation

New Technology: MS-based Proteomics

Genetically encoded phosphoserine and non-hydrolyzable phosphonate-containing phosphorylation mimics play a crucial role in studying phosphorylation-dependent processes. Genetic code expansion (GCE) allows the creation of site-specifically modified proteins with non-hydrolyzable and native phosphoserine. The precise engineering of phosphorylated proteins enables the measurement of phosphorylation-specific effects on protein function. Combining GCE with top-down mass spectrometry, enables an in-depth characterization of phospho-proteoforms which informs downstream binding studies. Here, we employ top-down analysis to characterize Bcl-XL, a protein central to cellular apoptosis. Within minutes of data collection, we performed sequence and impurity analysis on Bcl-XL variants containing phosphoserine and phospho mimics. These results underscore the importance of top-down characterization to inform studies on phosphorylation-dependent processes.Top-down analysis of Bcl-xL was performed using an Agilent 6545XT quadrupole time-of-flight (QTOF) mass spectrometer equipped with an ExD cell for electron fragmentation. Bcl-XL variants were expressed and purified by the GCE4All Center using amber codon reassignment to create pure, site-specifically modified Bcl-XL. Intact masses were determined using Mass Hunter BioConfirm. ExDViewer v4.5.11 was used for top-down sequence analysis and localization of modifications. Downstream binding studies were accomplished using isothermal titration calorimetry (ITC).In contrast to traditional bottom-up techniques that demand extensive sample preparation, our approach minimizes these requirements and reveals residue-level differences among samples within a 1-minute data collection period. We isolated the 19+ precursor of intact Bcl-XL proteins (~23kDa) with encoded phosphoserine, non-hydrolyzable phosphoserine, serine, glutamine, or alanine in position 62. Utilizing electron capture dissociation (ECD), we achieved 85-90% sequence coverage for all Bcl-XL variants including site-specific evidence of mutations and modifications. The spectrum included both ECD and collision-induced dissociation (CID) fragmentation ions which contributed to overall sequence coverage. For the wild-type Bcl-xL sample, the fragmentation results yielded 84% coverage considering ECD ions alone and 87% coverage when considering both ECD and CID-type ions. In addition, we show that the 2 Da difference between non-hydrolyzable phosphoserine with a phosphonate group and genuine phosphoserine can be differentiated using top-down electron capture mass spectrometry. The confirmation of modification identity &amp; site via top-down ECD revealed a contaminant in one phosphomimetic sample, leading to re-analysis of downstream binding studies using ITC. Effective analysis of unknown contaminants and precise mapping of modifications demonstrate the utility of pairing genetic code expansion with top-down analysis for phosphoprotein characterization.

Oral presentation

Prof.

High-throughput, single-cell top-down proteomics using multisegmented spray-capillary CE-MS

New Technology: Chromatography

Introduction: Ultrasensitive top-down proteomics techniques provide valuable insights into PTM-regulated cellular functions in mass-limited samples, including single cells. However, top-down proteomics analysis of mass-limited samples and single cells is challenging due to high sample complexity, wide dynamic range, and wide isotopic/charge state distributions of proteoforms. We recently developed &quot;Spray-Capillary&quot;, an ESI-assisted device for quantitative ultralow-volume sampling and online CE-MS analysis. In this study, we optimized the spray-capillary capillary device for improved sensitivity for intact protein analysis via PEI coating and reducing capillary inner diameter. We further coupled this platform droplet-based sample preparation to analyze single mammalian cells. Additionally, to improve throughput, we developed a multisegmented spray-capillary injection method that allows simultaneous separation of 10+ samples in a single analysis.Methods: Spray-capillary devices were fabricated as discussed previously (Huang et al. 2019). An ECE-001 CE autosampler (CMP scientific) was used for automation and a customized 3D stage was used for droplet sample injection. Data was collected on a Thermo Orbitrap Exploris 240 or Orbitrap Ascend Tribrid MS. Single cells were obtained from Hui Zhang at Johns Hopkins. Biopharma Finder and TopPIC Suite were utilized for mass feature deconvolution and identification, respectively.Results: We have developed and successfully applied spray-capillary CE-MS for quantitative single-cell metabolomics analysis in single onion cells (Huang et al. 2021). Here, we adapted this platform for automated, high-throughput single-cell top-down proteomics. First, we introduced a polyethyleneimine (PEI) coating to reduce sample loss due to protein adhesion and modulate EOF. The intensity for the coated capillary was 1.81E8 (RSD=17%), which represents a &gt;500-fold increase compared with the bare capillary. Additionally, 20 and 50 µm I.D. PEI-coated spray-capillary devices were evaluated, and the 20 µm capillary resulted in a ~32-fold higher BPE intensity compared with 50 μm. The optimized spray-capillary device was used to analyze 50 pg intact E. coli lysate and &gt;200 proteoforms were detected, representing one of the most sensitive detection methods for top-down proteomics. Additionally, we coupled a nanodroplet-based sample preparation method with this optimized spray-capillary CE-MS platform. We detected 867 and 711 intact proteoforms in ~45 HeLa and OVCAR-8 cells, respectively. We further applied this nanodroplet-based platform to single PC3 cells, detecting 60-80 proteoforms in single cells with high cell-to-cell reproducibility. Furthermore, to improve throughput, a multisegment sample injection method was coupled to our platform to improve throughput. Up to 17 sample segments could be injected and achieve baseline separation in a single spray-capillary CE-MS top-down proteomics analysis.

Spatially resolved multi-omics to decipher molecular pathology for idiopathic pulmonary fibrosis

Spatial and Imaging Proteomics

Introduction: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease characterized by progressive scarring of lung tissue, resulting in respiratory failure. Despite extensive research, the underlying molecular mechanisms driving IPF progression remain elusive. Here, we highlight the application of multi-omics spatial mass spectrometry tissue imaging coupled with laser capture microdissection (LCM)-LC-MS/MS technologies to identify key drivers of fibrosis progression, enabling target identification and therapeutic invention strategies for IPF.Methods: Sequential MALDI-MSI analysis was conducted on fresh-frozen IPF lung tissue sections for metabolite/lipid analysis and intact protein analysis using HiPlex-IHC (Ambergen) on a timsTOF fleX MALDI-2 (Bruker) instrument. SCiLS lab software (Bruker) was used for data visualization and analysis, while image segmentation algorithms embedded within QuPath and SCiLS lab were subsequently applied to enable the selection of regions of interest (ROI) for downstream analysis. LCM was performed using the LMD7 system (Leica) to isolate these ROIs for in-depth proteomic characterization via LC-MS/MS (timsTOF fleX MALDI-2 coupled with EvoSep).Preliminary data: Existing spatial omics studies in IPF have analyzed single omics layers, limiting their ability to capture the complex interplay between biomolecules. Our innovative approach utilizes sequential multi-omics MALDI imaging, which enables us to concurrently analyze and integrate information from untargeted metabolites and lipids alongside targeted proteins within a single tissue section. Our multi-omics analysis found dysregulation of inflammatory mediators, including increased expression of inflammatory metabolites like lysophosphatidic acid and arachidonic acid. These elevated levels coincide with the spatial distribution of CD68 macrophages, indicating a possible connection between these mediators and macrophage activity in the progression of IPF.Furthermore, our methodology incorporates laser capture microdissection (LCM) for targeted isolation of fibrotic regions, guided by a multi-modal approach. First, we utilize MALDI-IHC targeting α-SMA, an established protein marker for fibrosis, to identify regions of interest. The reliability of MALDI-IHC with α-SMA probes in determining fibrotic regions is then verified using pentachrome staining, a gold standard for characterizing fibrotic foci, ensuring that the most relevant areas for in-depth proteomic characterization via LC-MS/MS were isolated. Preliminary data from this approach indicates dysregulation in IPF-related pathways, including upregulated TGF-β signaling in fibrotic tissue, aligning with its role in ECM synthesis and remodeling. This localized analysis offers a deeper understanding of the molecular composition in fibrotic regions, overcoming limitations of bulk tissue analysis.

Conference room 3-4

Session

Top-down/middle-down proteomics

Session
Top-down/middle-down proteomics

Appointment

Chairs

Program

SessionTop-down/middle-down proteomics

Appointment

Chairs

Program

Session
Top-down/middle-down proteomics