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Reverse-phase protein arrays (RPPAs) have become an important tool for the sensitive and high-throughput detection of proteins from minute amounts of lysates from cell lines and cryopreserved tissue. The current standard method for tissue preservation in almost all hospitals worldwide is formalin fixation and paraffin embedding, and it would be highly desirable if RPPA could also be applied to formalin-fixed and paraffin embedded (FFPE) tissue. We investigated whether the analysis of FFPE tissue lysates with RPPA would result in biologically meaningful data in two independent studies. In the first study on breast cancer samples, we assessed whether a human epidermal growth factor receptor (HER) 2 score based on immunohistochemistry (IHC) could be reproduced with RPPA. The results showed very good concordance between the IHC and RPPA classifications of HER2 expression. In the second study, we profiled FFPE tumor specimens from patients with adenocarcinoma and squamous cell carcinoma in order to find new markers for differentiating these two subtypes of non-small cell lung cancer. p21-activated kinase 2 could be identified as a new differentiation marker for squamous cell carcinoma. Overall, the results demonstrate the technical feasibility and the merits of RPPA for protein expression profiling in FFPE tissue lysates.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3769334 | PMC |
http://dx.doi.org/10.1074/mcp.M112.023051 | DOI Listing |
The ability to bring spatial resolution to omics studies enables a deeper understanding of cell populations and interactions in biological tissues. In the case of proteomics, single-cell and spatial approaches have been particularly challenging, due to limitations in sensitivity and throughput relative to other omics fields. Recent developments at the level of sample handling, chromatography, and mass spectrometry have set the stage for proteomics to be established in these new disciplines.
View Article and Find Full Text PDFMethods Mol Biol
December 2024
Department of Pharmacology & Immunology, Medical University of South Carolina, Charleston, SC, USA.
Recent work in single-cell imaging has allowed unprecedented insight into single-cell interactions that control disease progression. However, approaches to understanding the combined extracellular and cellular microenvironment are limited. In the current protocol, we describe an approach that allows single-cell type imaging using matrix-assisted laser desorption/ionization immunohistochemistry (MALDI-IHC) of UV (ultraviolet) photocleavable mass tags combined with N-glycomic and ECM-targeted proteomic imaging from the same formalin-fixed paraffin-embedded tissue section.
View Article and Find Full Text PDFNPJ Precis Oncol
December 2024
Eye Center, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China.
Eyelid tumors pose diagnostic challenges due to their diverse pathological types and limited biopsy materials. This study aimed to develop an artificial intelligence (AI) diagnostic system for accurate classification of eyelid tumors. Utilizing mass spectrometry-based proteomics, we analyzed proteomic data from eight tissue types and identified eighteen novel biomarkers based on 233 formalin-fixed, paraffin-embedded (FFPE) samples from 150 patients.
View Article and Find Full Text PDFSpatially mapping the transcriptome and proteome in the same tissue section can significantly advance our understanding of heterogeneous cellular processes and connect cell type to function. Here, we present Deterministic Barcoding in Tissue sequencing plus (DBiTplus), an integrative multi-modality spatial omics approach that combines sequencing-based spatial transcriptomics and image-based spatial protein profiling on the same tissue section to enable both single-cell resolution cell typing and genome-scale interrogation of biological pathways. DBiTplus begins with reverse transcription for cDNA synthesis, microfluidic delivery of DNA oligos for spatial barcoding, retrieval of barcoded cDNA using RNaseH, an enzyme that selectively degrades RNA in an RNA-DNA hybrid, preserving the intact tissue section for high-plex protein imaging with CODEX.
View Article and Find Full Text PDFF S Sci
December 2024
Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Northwestern University Feinberg School of Medicine, 259 E Erie St Suite 2400, Chicago, IL 60611, USA; Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Warren Alpert Medical School of Brown University, 101 Dudley St., Providence, RI 02905, USA.
Objective: To evaluate the uterine microbiome among women with endometrial polyps and submucosal fibroids and to compare results between endometrial sampling techniques.
Design: Patients with polyps or fibroids were prospectively recruited prior to hysteroscopy, while patients undergoing retrieval for planned oocyte cryopreservation were recruited prospectively as controls. Three specimen types obtained for each patient were the distal 5 mm of an embryo catheter passed to the uterine fundus (C), endometrial tissue from an endometrial biopsy (T), and formalin fixed paraffin embedded endometrial tissue from the same endometrial biopsy (FFPE).
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