Beads-free protein immunoprecipitation for a mass spectrometry-based interactome and posttranslational modifications analysis

Proteome Science

Methodology

Michal Mikula¹  Malgorzata Statkiewicz¹  Jakub Karczmarski¹  Jerzy Ostrowski²  Karol Bomsztyk³  Tymon Rubel⁴ 

[1] Department of Genetics, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, 02-781, Warsaw, Poland;Department of Genetics, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, 02-781, Warsaw, Poland;Department of Gastroenterology, Hepatology and Clinical Oncology, Medical Center for Postgraduate Education, 01-813, Warsaw, Poland;Department of Medicine, University of Washington, 98109, Seattle, WA, USA;Institute of Radioelectronics, Warsaw University of Technology, 00-665, Warsaw, Poland;

关键词: hnRNP K;    Immuoprecipitation;    Interactome;    Post-translational modifications;    Quantitative proteomics;   

DOI  :  10.1186/s12953-015-0079-0

 received in 2015-04-16, accepted in 2015-08-20,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundProtein immunoprecipitation (IP) coupled with MS provides means to interrogate protein complexes and their posttranslational modifications (PTMs). In a typical protein IP assay antibodies are conjugated to protein A/G beads requiring large amounts of antibodies, tube transfers and centrifugations.ResultsAs an alternative, we present Matrix-IP, beads-free microplate-based platform with surface-immobilized antibodies. Assay utilizes standard 96-well polypropylene PCR plates that are laboratory-fabricated with UV-C light and then protein A/G coated prior to IP reaction. We demonstrate application of Matrix-IP platform in MS analysis of heterogeneous nuclear ribonucleoprotein K (hnRNP K) interactome and PTMs.ConclusionMatrix-IP is time-saving, easy to use high throughput method adaptable for low sample amounts and automation.

【 授权许可】

CC BY   
© Mikula et al. 2015