Chung, C.-W. | bioRxiv Science

bioRxiv2023

Microscopy-guided subcellular proteomic discovery by high-speed ultra-content photo-biotinylation

Microscopy-guided proteomics at an organelle-dimension resolution is desired for revealing unknown protein constituents at specific disease- or functional-associated regions at the molecular-molecular interactions level. Here, we achieve protein spatial purification by introducing a firmware-integrated microscopy platform that triggers in situ subcellular photo-biotinylation of proteins at user-defined regions of interest (ROIs) one field of view (FOV) at a time for thousands of FOVs fully automatically. An illumination pattern at the analogous ROIs of each FOV is calculated on the fly by either machine learning or traditional image processing. Photoactivatable amino acid crosslinkers are activated by a two-photon focal light one spot at a time at a sub-millisecond illumination duration per spot. Imaging, pattern generation, targeted illumination, and FOV movement are coordinated and cycled with high-speed mechatronic control to complete illumination on millions of ROI spots within hours. Once enough proteins are biotinylated in a cell or tissue sample, the sample is scraped and lysed, and avidin pulldown is used to enrich proteins to achieve spatial protein scooping at a 240-nm precision. Subsequent LC-MS/MS is implemented to reveal the subcellular proteome in high sensitivity, specificity, and resolution. Using this technology termed optoproteomics, we have revealed novel stress granule-localized and amyloid {beta}-localized proteins validated by immunostaining. Together, spatial purification by ultra-content, high-speed microscopy-targeted photo-biotinylation enables unprecedented subcellular spatial proteomics discovery in any microscopically recognizable regions.

cell biology↗

bioRxiv2019

Significantly different expression levels of microRNAs associated with vascular invasion in hepatocellular carcinoma and their prognostic significance after surgical resection

BackgroundAlthough gross vascular invasion (VI) has prognostic significance in patients with hepatocellular carcinoma (HCC) who have undergone hepatic resection, few studies have investigated the relationship between gross VI and aberrant expression of microribonucleic acids (miRNAs and miRs). Thus, the objective of this study was to identify miRNAs selectively expressed in HCC with gross VI and investigate their prognostic significance.\n\nMaterials and MethodsEligible two datasets (accession number: GSE20594 and GSE67140) were collected from the National Center for Biotechnology Informations (NCBI) Gene Expression Omnibus (GEO) database to compare miRNAs expression between HCC with and without gross VI. Differentially expressed miRNAs were externally validated using expression data from The Cancer Genome Atlas (TCGA) database. Prognostic significance and predicted functions of selected miRNAs for HCC were also investigated.\n\nResultsThirty-five miRNAs were differentially expressed between HCC with and without gross VI in both datasets. Among them, four miRNAs were validated using TCGA database. miR-582 was upregulated to a greater extent while miR-99a, miR-100, and miR-148a were downregulated to a greater extent in patients with HCC and gross VI than in those with HCC but no VI. Receiver operating characteristic (ROC) curve analysis showed discriminatory power of these miRNAs in predicting gross VI. Multivariate survival analysis revealed that types of surgery, advanced tumor node metastasis (TNM) stage, and miR-100 underexpression were independently associated with tumor recurrence. It also revealed that types of surgery, advanced TNM stage, miR-100 underexpression, and miR-582 overexpression were independent risk factors for overall survival (OS) after hepatic resection for HCC. A text mining analysis revealed that these miRNAs were linked to multifaceted hallmarks of cancer, including \"invasion and metastasis.\"\n\nConclusionsmiR-100 underexpression and miR-582 overexpression were associated with gross VI and poor survival of patients after hepatic resection for HCC.

cancer biology↗