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

Proteome Science
Michał MikulaJerzy Ostrowski

Abstract

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. 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. Matrix-IP is time-saving, easy to use high throughput method adaptable for low sample amounts and automation.

References

May 18, 1999·The Journal of Biological Chemistry·D S SchulleryK Bomsztyk
Apr 29, 2004·Analytical Biochemistry·Surbhi Desai, Boguslawa R Dworecki
Jun 2, 2004·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·Karol BomsztykJerzy Ostrowski
Mar 7, 2006·Proteomics·Michał MikulaJerzy Ostrowski
Dec 11, 2007·Journal of Proteome Research·Lukas KällWilliam Stafford Noble
Mar 29, 2008·Methods in Molecular Biology·Barbara Kaboord, Maria Perr
May 6, 2008·Bioinformatics·Ashoka D PolpitiyaRichard D Smith
Jul 1, 2010·DNA Research : an International Journal for Rapid Publication of Reports on Genes and Genomes·Michal MikulaJerzy Ostrowski
Nov 15, 2011·Journal of Proteome Research·Thomas TausKarl Mechtler
Jul 25, 2012·The Journal of Biological Chemistry·Federico PelischAnabella Srebrow
Dec 4, 2012·Nucleic Acids Research·Andrea FranceschiniLars J Jensen
Jul 6, 2014·Clinical Proteomics·Jakub KarczmarskiJerzy Ostrowski
Dec 18, 2014·Nucleic Acids Research·Peter V HornbeckElzbieta Skrzypek

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Citations

Jul 18, 2017·International Journal of Oncology·Mateusz BujkoJanusz Aleksander Siedlecki
Mar 14, 2017·Frontiers in Molecular Neuroscience·Sanjeev V Namjoshi, Kimberly F Raab-Graham

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Methods Mentioned

BETA
immunoprecipitation
acetylations
co-IPs
PCR
ChIP
RIP
electrophoresis

Software Mentioned

Mascot Distiller
Matrix IP
Mascot
MS
CLC Sequence Viewer
MScan

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