Project description:The molecular identification of tissue proteoforms by top-down mass spectrometry (TDMS) is significantly limited by throughput and dynamic range. We introduce AutoPiMS, a single-ion MS based multiplexed workflow for top-down tandem MS (MS2) directly from tissue microenvironments in a semi-automated manner. AutoPiMS directly off human ovarian cancer sections allowed for MS2 identification of 73 proteoforms up to 54 kDa at a rate of <1 min per proteoform. AutoPiMS is directly interfaced with multifaceted proteoform imaging MS data modalities for the identification of proteoform signatures in tumor and stromal regions in ovarian cancer biopsies. From a total of ~1000 proteoforms detected by region-of-interest label-free quantitation, we discover 303 differential proteoforms in stroma versus tumor from the same patient. 14 of the top proteoform signatures are corroborated by MSI at 20 micron resolution including the differential localization of methylated forms of CRIP1, indicating the importance of proteoform-enabled spatial biology in ovarian cancer.
Project description:Spatially resolved characterization of proteoforms has great potential to significantly advance our understanding of physiological and disease mechanisms. However, it faces challenges regarding throughput and coverage. Herein, we developed a robust method for high-throughput proteoform imaging (HTPi) by combining matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) and region-specific top-down proteomic analysis.
Project description:Mass spectrometry (MS)-based spatially resolved top-down proteomics (TDP) of tissues is crucial for understanding the roles played by microenvironmental heterogeneity in the biological functions of organs and for discovering new proteoform biomarkers of diseases. There are few published spatially resolved TDP studies. One of the challenges relates to the limited performance of TDP for the analysis of spatially isolated samples using, for example, laser capture microdissection (LCM) because those samples are usually mass-limited. We present the first pilot study of LCM-capillary zone electrophoresis (CZE)-MS/MS for spatially resolved TDP and used zebrafish brain as the sample. The LCM-CZE-MS/MS platform employed a non-ionic detergent and a freeze-thaw method for efficient proteoform extraction from LCM isolated brain sections followed by CZE-MS/MS without any sample cleanup step, ensuring high sensitivity. Over 400 proteoforms were identified in a CZE-MS/MS analysis of one LCM brain section via consuming the protein content of roughly 250 cells. We observed drastic differences in proteoform profiles between two LCM brain sections isolated from the optic tectum (Teo) and telencephalon (Tel) regions. Proteoforms of three proteins (npy, penkb, and pyya) having neuropeptide hormone activity were exclusively identified in the isolated Tel section. Proteoforms of reticulon, myosin, and troponin were almost exclusively identified in the isolated Teo section, and those proteins play essential roles in visual and motor activities. The proteoform profiles accurately reflected the main biological functions of the Teo and Tel regions of the brain. Additionally, hundreds of post-translationally modified proteoforms were identified.
Project description:DBiTplus utilizes in situ reverse transcription, in-tissue delivery of DNA oligos for spatial barcoding, and RNaseH-mediated selective cDNA retrieval, preserving tissue architecture to enable highly multiplexed protein imaging and spatial transcriptomics on the same slide. Computational pipelines seamlessly integrates these modalities, allowing imaging-guided deconvolution to generate genome-scale, single-cell-resolved spatial transcriptome atlases. DBiTplus was demonstrated across diverse samples including mouse embryos, normal human lymph nodes, and formalin-fixed paraffin-embedded (FFPE) human lymphoma tissues, highlighting its compatibility with challenging clinical specimens. Applying DBiTplus to human lymphoma samples reveals key mechanisms driving lymphomagenesis, progression, and notably, the progression of indolent marginal zone lymphoma (MZL) or the transformation from chronic lymphocytic leukemia (CLL) to diffuse large B-cell lymphoma (DLBCL), including uniquely, the spatially resolved involvement of microRNAs in modulating the transformation dynamics. Thus, DBiTplus is a unified workflow including integrative experimental procedure and computational innovation for spatially resolved single-cell atlasing and exploration of biological pathways cell-by-cell at genome-scale.
Project description:The main goal of this study is to explore the proteomic expression in prophylactic salpingooophorectomy specimens, obtained from highly selected cohort of patients at risk of developing a high-grade serous ovarian carcinoma (HGSOC), because of a hereditary (BRCA 1 or 2 mutation) or a documented familial context. Pathological aspects of fallopian tube specimens, at the origin of most HGSOC in this selected feminine population, are extracted from slides annotated by the pathologist, then submitted to a proteomic analysis. We carried out an in-depth proteomics analysis of these epithelial lesions (p53 signature, serous tubal intraepithelial carcinoma-STIC and serous tubal intraepithelial lesions-STIL) based on spatially resolved proteomic guided by IHC technique.
Project description:Here we present an approach to identify N-linked glycoproteins and deduce their spatial localization using a combination of MALDI N-glycan MSI and spatially-resolved glycoproteomics. We subjected glioma biopsies to on-tissue PNGaseF digestion and MALDI-MSI and found that the glycan HexNAc4-Hex5-NeuAc2 was predominantly expressed in necrotic regions of high-grade canine gliomas. To determine the underlying sialo-glycoprotein, various regions in adjacent tissue sections were subjected to microdigestion and manual glycoproteomic analysis. Results identified haptoglobin as the protein associated with HexNAc4-Hex5-NeuAc2, making our study the first report that directly links glycan imaging with intact glycopeptide identification. In total, our spatially-resolved glycoproteomics technique identified over 400 N-, O-, and S- glycopeptides from over 30 proteins, demonstrating the diverse array of glycosylation present on the tissue slides and the sensitivity of our technique. Ultimately, this proof-of-principle work demonstrates that spatially-resolved glycoproteomics greatly complement MALDI-MSI in understanding dysregulated glycosylation.
Project description:Here we present an approach to identify N-linked glycoproteins and deduce their spatial localization using a combination of MALDI N-glycan MSI and spatially-resolved glycoproteomics. We subjected glioma biopsies to on-tissue PNGaseF digestion and MALDI-MSI and found that the glycan HexNAc4-Hex5-NeuAc2 was predominantly expressed in necrotic regions of high-grade canine gliomas. To determine the underlying sialo-glycoprotein, various regions in adjacent tissue sections were subjected to microdigestion and manual glycoproteomic analysis. Results identified haptoglobin as the protein associated with HexNAc4-Hex5-NeuAc2, making our study the first report that directly links glycan imaging with intact glycopeptide identification. In total, our spatially-resolved glycoproteomics technique identified over 400 N-, O-, and S- glycopeptides from over 30 proteins, demonstrating the diverse array of glycosylation present on the tissue slides and the sensitivity of our technique. Ultimately, this proof-of-principle work demonstrates that spatially-resolved glycoproteomics greatly complement MALDI-MSI in understanding dysregulated glycosylation.
Project description:Method development for high-resolution spatially-resolved proteome mapping of tissue heterogeneity. Laser microdissection was coupled with nanodroplet sample preparation and ultrasensitive LC-MS.
Project description:Advancing our understanding of embryonic development is heavily dependent on identification of novel pathways or regulators. While genome-wide techniques such as RNA sequencing are ideally suited for discovering novel candidate genes, they are unable to yield spatially resolved information in embryos or tissues. Microscopy-based approaches, using for example in situ hybridization, can provide spatial information about gene expression, but are limited to analyzing one or a few genes at a time. Here, we present a method where we combine traditional histological techniques with low-input RNA sequencing and mathematical image reconstruction to generate a high-resolution genome-wide 3D atlas of gene expression in the zebrafish embryo at three developmental stages. We also demonstrate that our technique is suitable for spatially-resolved differential expression analysis in wildtype and Gli3 mutant mouse forelimbs. Importantly, our method enables searching for genes that are expressed in specific spatial patterns without manual image annotation. We envision broad applicability of RNA tomography as an accurate and sensitive approach for spatially resolved transcriptomics in whole embryos and dissected organs. To generate spatially-resolved RNA-seq data for zebrafish embryos (shield stage, 10 somites, 15 somites, 18 somites) and mouse forelimbs (E10.5), we cryosectioned samples, extracted RNA from the individual sections, and amplified and barcoded mRNA using the CEL-seq protocol (Hashimshony et al., Cell Reports, 2012) with a few modifications. Libraries were sequenced on Illumina HiSeq 2500 using 50bp paired end sequencing. Selected zebrafish libraries were sequenced on MiSeq 250bp paired-end to improve 3' annotations.