Detection of post-translational modifications using solid-phase proximity ligation assay

Felipe Marques Souza de Oliveira; Stefan Mereiter; Peter Lönn; Benjamin Siart; Qiujin Shen; Johan Heldin; Doroteya Raykova; Niclas G Karlsson; Karol Polom; Franco Roviello; Celso A Reis; Masood Kamali-Moghaddam

doi:10.1016/j.nbt.2017.10.005

Detection of post-translational modifications using solid-phase proximity ligation assay

N Biotechnol. 2018 Oct 25:45:51-59. doi: 10.1016/j.nbt.2017.10.005. Epub 2017 Oct 31.

Affiliations

¹ Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
² i3S - Instituto de Investigação e Inovação em Saúde and IPATIMUP - Institute of Molecular Pathology and Immunology of the University of Porto, Portugal.
³ Department of Anthropology, University of Vienna, Austria; Department of Behavioral Biology, University of Vienna, Austria.
⁴ Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden; Department of Pharmaceutical Biosciences, Uppsala University, Sweden.
⁵ Department of Medical Biochemistry and Cell Biology at Institute of Biomedicine, Gothenburg University, Sweden.
⁶ Department of Surgical Oncology, Medical University of Gdansk, Poland; General Surgery and Surgical Oncology Department, Università deli Studi di Siena, Italy.
⁷ General Surgery and Surgical Oncology Department, Università deli Studi di Siena, Italy.
⁸ i3S - Instituto de Investigação e Inovação em Saúde and IPATIMUP - Institute of Molecular Pathology and Immunology of the University of Porto, Portugal; Instituto de Ciências Biomédicas Abel Salazar (ICBAS), University of Porto, Portugal; Faculty of Medicine of the University of Porto, Portugal.
⁹ Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden. Electronic address: masood.kamali@igp.uu.se.

PMID: 29101055
DOI: 10.1016/j.nbt.2017.10.005

Abstract

Post-translational modifications (PTMs) regulate protein activities to help orchestrate and fine-tune cellular processes. Dysregulation of PTMs is often related with disorders and malignancies, and may serve as a precise biomarker of disease. Developing sensitive tools to measure and monitor low-abundant PTMs in tissue lysates or serum will be instrumental for opening up new PTM-based diagnostic avenues. Here, we investigate the use of solid-phase proximity ligation assay (SP-PLA) for detection of different PTMs. The assay depends on the recognition of the target protein molecule and its modification by three affinity binders. Using antibodies and lectins, we applied the method for detection of glycosylated CD44 and E-Cadherin, and phosphorylated p53 and EGFR. The assay was found to have superior dynamic range and limit of detection compared to standard ELISAs. In summary, we have established the use of SP-PLA as an appropriate method for sensitive detection of PTMs in lysates and sera, which may provide a basis for future PTM-based diagnostic and prognostic biomarkers.

Keywords: CD44; E-cadherin; EGFR; Gastric cancer; Glycosylation; Phosphorylation; Post-translational modifications; Protein detection; Solid-phase proximity ligation assay (SP-PLA); p53.

MeSH terms

Aged
Cell Line
ErbB Receptors / analysis
ErbB Receptors / metabolism*
Female
Humans
Hyaluronan Receptors / analysis
Hyaluronan Receptors / metabolism
Immunoassay*
Male
Protein Processing, Post-Translational
Stomach Neoplasms / diagnosis
Stomach Neoplasms / metabolism*
Tumor Suppressor Protein p53 / analysis
Tumor Suppressor Protein p53 / metabolism*

Substances

Hyaluronan Receptors
Tumor Suppressor Protein p53
ErbB Receptors