Dataset Information

One-STAGE Tip Method for TMT-Based Proteomic Analysis of a Minimal Amount of Cells.

ABSTRACT: Liquid chromatography-tandem mass spectrometry (LC-MS)-based profiling of proteomes with isobaric tag labeling from low-quantity biological and clinical samples, including needle-core biopsies and laser capture microdissection, has been challenging due to the limited amount and sample loss during preparation. To address this problem, we developed OnM (On-Column from Myers et al. and mPOP)-modified on-column method combining freeze-thaw lysis of mPOP with isobaric tag labeling of On-Column method to minimize sample loss. OnM is a method that processes the sample in one-STAGE tip from cell lysis to tandem mass tag (TMT) labeling without any transfer of the sample. In terms of protein coverage, cellular components, and TMT labeling efficiency, the modified On-Column (or OnM) displayed similar performance to the results from Myers et al. To evaluate the lower-limit processing capability of OnM, we utilized OnM for multiplexing and were able to quantify 301 proteins in a TMT 9-plex with 50 cells per channel. We optimized the method as low as 5 cells per channel in which we identified 51 quantifiable proteins. OnM method is a low-input proteomics method widely applicable and capable of identifying and quantifying proteomes from limited samples, with tools that are readily available in a majority of proteomic laboratories.

SUBMITTER: Park N

PROVIDER: S-EPMC10249390 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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One-STAGE Tip Method for TMT-Based Proteomic Analysis of a Minimal Amount of Cells.

Park Narae N Lee Hankyul H Kwon Yumi Y Ju Shinyeong S Lee Seonjeong S Yoo Seongjin S Park Kang-Sik KS Lee Cheolju C

ACS omega 20230524 22

Liquid chromatography-tandem mass spectrometry (LC-MS)-based profiling of proteomes with isobaric tag labeling from low-quantity biological and clinical samples, including needle-core biopsies and laser capture microdissection, has been challenging due to the limited amount and sample loss during preparation. To address this problem, we developed OnM (On-Column from Myers et al. and mPOP)-modified on-column method combining freeze-thaw lysis of mPOP with isobaric tag labeling of On-Column method ...[more]

PMID: 37305273

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Project description:BackgroundCardiovascular diseases remain the leading cause of morbidity and mortality worldwide, most of which are caused by atherosclerosis. Discerning processes that participate in macrophage-to-foam cell formation are critical for understanding the basic mechanisms underlying atherosclerosis. To explore the molecular mechanisms of foam cell formation, differentially expressed proteins were identified.MethodsHuman peripheral blood mononuclear cells were stimulated with macrophage colony-stimulating factor, and obtained macrophages were transformed into foam cells by oxidized low-density lipoprotein. Tandem mass tag (TMT) labeling combined with mass spectrometry was performed to find associations between foam cell transformation and proteome profiles.ResultsTotally, 5146 quantifiable proteins were identified, among which 1515 and 182 differentially expressed proteins (DEPs) were found in macrophage/monocyte and foam cell/macrophage, respectively. Subcellular localization analysis revealed that downregulated DEPs of macrophages/monocytes were mostly located in the nucleus, whereas upregulated DEPs of foam cells/macrophages were mostly extracellular or located in the plasma membrane. Functional analysis of DEPs demonstrated that cholesterol metabolism-related proteins were upregulated in foam cells, whereas immune response-related proteins were downregulated in foam cells. The protein interaction network showed that the DEPs with the highest interaction scores between macrophages and foam cells were mainly concentrated in lysosomes and the endoplasmic reticulum.ConclusionsProteomics analysis suggested that cholesterol metabolism was upregulated, while the immune response was suppressed in foam cells. KEGG enrichment analysis and protein-protein interaction analysis indicated that DEPs located in the endoplasmic reticulum and lysosomes might be key drivers of foam cell formation. These data provide a basis for identifying the potential proteins associated with the molecular mechanism underlying macrophage transformation to foam cells.

Dataset Information

One-STAGE Tip Method for TMT-Based Proteomic Analysis of a Minimal Amount of Cells.

Publications

One-STAGE Tip Method for TMT-Based Proteomic Analysis of a Minimal Amount of Cells.

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