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An optimized co-immunoprecipitation protocol for the analysis of endogenous protein-protein interactions in cell lines using mass spectrometry.

ABSTRACT: This protocol represents an optimized proteomics-based protocol for the endogenous protein enrichment and protein-protein interaction analysis. This 2-step protocol consists of: 1) co-immunoprecipitation of the bait protein; 2) the bait-protein interactions analysis using LC-MS/MS. Here, we used Dynabeads® for the enrichment of the target protein (the bait) and its interactors. We have tested the protocol using several different cell lines. Our conclusion is that the protocol is applicable to different cell lines and species. For complete details on the use and execution of this protocol, please refer to Lagundžin et al. (2019).

SUBMITTER: Lagundzin D

PROVIDER: S-EPMC8920916 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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An optimized co-immunoprecipitation protocol for the analysis of endogenous protein-protein interactions in cell lines using mass spectrometry.

Lagundžin Dragana D Krieger Kimiko L KL Law Henry C-H HC Woods Nicholas T NT

STAR protocols 20220310 1

This protocol represents an optimized proteomics-based protocol for the endogenous protein enrichment and protein-protein interaction analysis. This 2-step protocol consists of: 1) co-immunoprecipitation of the bait protein; 2) the bait-protein interactions analysis using LC-MS/MS. Here, we used Dynabeads® for the enrichment of the target protein (the bait) and its interactors. We have tested the protocol using several different cell lines. Our conclusion is that the protocol is applicable to di ...[more]

PMID: 35300004

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Project description:Gangliosides are glycosphingolipids composed of a sialylated glycan head group and a ceramide backbone. These anionic lipids form lipid rafts and play crucial roles in regulating various proteins involved in signal transduction, adhesion, and cell-cell recognition. Neuroblastoma, a pediatric cancer of the sympathetic nervous system, is treated with intensive chemotherapy, radiation, and an antibody targeting the GD2 ganglioside. Gangliosides are critical in neuroblastoma development and serve as therapeutic targets, making it essential to establish a reliable, rapid, and cost-effective method for profiling gangliosides, particularly one capable of isomeric separation of intact species. In this study, liquid chromatography-mass spectrometry (LC-MS) was optimized using standard gangliosides, followed by the optimization of sphingolipid extraction methods from cell lines by comparing Folch and absolute methanol extraction techniques. Percent recovery and the number of identified sphingolipids were used to evaluate the analytical merits of these methods. A standard gangliosides calibration curve demonstrated excellent linearity (R2 = 0.9961-0.9975). The ZIC-HILIC column provided the best separation of ganglioside GD1 isomers with a 25 min runtime. GD1a elutes before GD1b on the ZIC-HILIC column. Absolute methanol yielded better percent recovery (96 ± 7) and identified 121 different sphingolipids, the highest number between the two extraction methods. The optimized method was applied to profile gangliosides in neuroblastoma (COG-N-683), pancreatic cancer (PSN1), breast cancer (MDA-MB-231BR), and brain tumor (CRL-1620) cell lines. The ganglioside profile of the neuroblastoma cell line COG-N-683 showed an inverse relationship between GD1 and GD2. Ceramide, Hex1Cer, GM1, and GM3 were highly abundant in CRL-1620, PSN1, and MDA-MB-231BR, respectively. These results suggest that our method provides a sensitive, reliable, and high-throughput workflow for ganglioside profiling across different cell types.

Dataset Information

An optimized co-immunoprecipitation protocol for the analysis of endogenous protein-protein interactions in cell lines using mass spectrometry.

Publications

An optimized co-immunoprecipitation protocol for the analysis of endogenous protein-protein interactions in cell lines using mass spectrometry.

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