{"title":"两步富集法有助于降低溶酶体蛋白质组分析的背景。","authors":"Sara Bonini, Dominic Winter","doi":"10.1021/acs.jproteome.4c00053","DOIUrl":null,"url":null,"abstract":"<p><p>Lysosomes constitute the main degradative compartment of most mammalian cells and are involved in various cellular functions. Most of them are catalyzed by lysosomal proteins, which typically are low abundant, complicating their analysis by mass spectrometry-based proteomics. To increase analytical performance and to enable profiling of lysosomal content, lysosomes are often enriched. Two approaches have gained popularity in recent years, namely, superparamagnetic iron oxide nanoparticles (SPIONs) and immunoprecipitation from cells overexpressing a 3xHA-tagged version of TMEM192 (TMEM-IP). The effect of these approaches on the lysosomal proteome has not been investigated to date. We addressed this topic through a combination of both techniques and proteomic analysis of lysosome-enriched fractions. For SPIONs treatment, we identified altered cellular iron homeostasis and moderate changes of the lysosomal proteome. For overexpression of TMEM192, we observed more pronounced effects in lysosomal protein expression, especially for lysosomal membrane proteins and those involved in protein trafficking. Furthermore, we established a combined strategy based on the sequential enrichment of lysosomes with SPIONs and TMEM-IP. This enabled increased purity of lysosome-enriched fractions and, through TMEM-IP-based lysosome enrichment from SPIONs flow-through and eluate fractions, additional insights into the properties of individual approaches. All data are available via ProteomeXchange with PXD048696.</p>","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":null,"pages":null},"PeriodicalIF":3.8000,"publicationDate":"2024-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Two-Step Enrichment Facilitates Background Reduction for Proteomic Analysis of Lysosomes.\",\"authors\":\"Sara Bonini, Dominic Winter\",\"doi\":\"10.1021/acs.jproteome.4c00053\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Lysosomes constitute the main degradative compartment of most mammalian cells and are involved in various cellular functions. Most of them are catalyzed by lysosomal proteins, which typically are low abundant, complicating their analysis by mass spectrometry-based proteomics. To increase analytical performance and to enable profiling of lysosomal content, lysosomes are often enriched. Two approaches have gained popularity in recent years, namely, superparamagnetic iron oxide nanoparticles (SPIONs) and immunoprecipitation from cells overexpressing a 3xHA-tagged version of TMEM192 (TMEM-IP). The effect of these approaches on the lysosomal proteome has not been investigated to date. We addressed this topic through a combination of both techniques and proteomic analysis of lysosome-enriched fractions. For SPIONs treatment, we identified altered cellular iron homeostasis and moderate changes of the lysosomal proteome. For overexpression of TMEM192, we observed more pronounced effects in lysosomal protein expression, especially for lysosomal membrane proteins and those involved in protein trafficking. Furthermore, we established a combined strategy based on the sequential enrichment of lysosomes with SPIONs and TMEM-IP. This enabled increased purity of lysosome-enriched fractions and, through TMEM-IP-based lysosome enrichment from SPIONs flow-through and eluate fractions, additional insights into the properties of individual approaches. All data are available via ProteomeXchange with PXD048696.</p>\",\"PeriodicalId\":48,\"journal\":{\"name\":\"Journal of Proteome Research\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.8000,\"publicationDate\":\"2024-07-05\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Proteome Research\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1021/acs.jproteome.4c00053\",\"RegionNum\":2,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Proteome Research","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1021/acs.jproteome.4c00053","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
Two-Step Enrichment Facilitates Background Reduction for Proteomic Analysis of Lysosomes.
Lysosomes constitute the main degradative compartment of most mammalian cells and are involved in various cellular functions. Most of them are catalyzed by lysosomal proteins, which typically are low abundant, complicating their analysis by mass spectrometry-based proteomics. To increase analytical performance and to enable profiling of lysosomal content, lysosomes are often enriched. Two approaches have gained popularity in recent years, namely, superparamagnetic iron oxide nanoparticles (SPIONs) and immunoprecipitation from cells overexpressing a 3xHA-tagged version of TMEM192 (TMEM-IP). The effect of these approaches on the lysosomal proteome has not been investigated to date. We addressed this topic through a combination of both techniques and proteomic analysis of lysosome-enriched fractions. For SPIONs treatment, we identified altered cellular iron homeostasis and moderate changes of the lysosomal proteome. For overexpression of TMEM192, we observed more pronounced effects in lysosomal protein expression, especially for lysosomal membrane proteins and those involved in protein trafficking. Furthermore, we established a combined strategy based on the sequential enrichment of lysosomes with SPIONs and TMEM-IP. This enabled increased purity of lysosome-enriched fractions and, through TMEM-IP-based lysosome enrichment from SPIONs flow-through and eluate fractions, additional insights into the properties of individual approaches. All data are available via ProteomeXchange with PXD048696.
期刊介绍:
Journal of Proteome Research publishes content encompassing all aspects of global protein analysis and function, including the dynamic aspects of genomics, spatio-temporal proteomics, metabonomics and metabolomics, clinical and agricultural proteomics, as well as advances in methodology including bioinformatics. The theme and emphasis is on a multidisciplinary approach to the life sciences through the synergy between the different types of "omics".