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


RESULTS
As 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.


CONCLUSIONS
Matrix-IP is time-saving, easy to use high throughput method adaptable for low sample amounts and automation.
Department of Genetics, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, 02-781 Warsaw, Poland.
Michal
M
Mikula
Institute of Radioelectronics, Warsaw University of Technology, 00-665 Warsaw, Poland.
Tymon
T
Rubel
Department of Genetics, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, 02-781 Warsaw, Poland.
Jakub
J
Karczmarski
Department of Genetics, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, 02-781 Warsaw, Poland.
Malgorzata
M
Statkiewicz
Department of Medicine, University of Washington, Seattle, 98109 WA USA.
Karol
K
Bomsztyk
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.
Jerzy
J
Ostrowski
Immuoprecipitation; Interactome; Post-translational modifications; Quantitative proteomics; hnRNP K