Phosphotyrosine Proteomic Study of Interferon α Signaling Pathway Using a Combination of Immunoprecipitation and Immobilized Metal Affinity Chromatography

Haiyan Zheng,Ping Hu,Douglas F Quinn,Y Karen Wang

doi:10.1074/mcp.m400077-mcp200

Haiyan Zheng, Ping Hu + Show 2 more

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https://doi.org/10.1074/mcp.m400077-mcp200

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Abstract

Tyrosine phosphorylation is a type of post-translational modification that plays a crucial role in signal transduction. Thus, the study of this modification at the proteomic level has great biological significance. However, because of the low abundance of tyrosine-phosphorylated proteins in total cell lysate, it is difficult to evaluate the dynamics of tyrosine phosphorylation at a global level. In this work, proteins carrying phosphotyrosine (pTyr) were first purified from whole cell lysate by immunoprecipitation using anti-pTyr monoclonal antibodies. After tryptic digestion, phosphopeptides were further enriched by IMAC and analyzed by LC-MS. Quantitative changes of tyrosine phosphorylation at the global level were evaluated using isotopic labeling (introduced at the methyl esterification step prior to IMAC). Using this double enrichment approach, we characterized interferon alpha (IFNalpha)-induced pTyr proteomic changes in Jurkat cells. We observed induced phosphorylation on several well documented as well as novel tyrosine phosphorylation sites on proteins involved in IFNalpha signal transduction, such as Tyk2, JAK1, and IFNAR subunits. A specific site on alpha-tubulin (Tyr-271) was observed to be phosphorylated upon treatment as well. Furthermore, our results suggest that LOC257106, a CDC42 GAP-like protein, is potentially involved in this pathway.

Highlights

Tyrosine phosphorylation is a type of post-translational modification that plays a crucial role in signal transduction
Using STAT1 as a marker, we found that the treatment of Jurkat cells with IFN␣ at a concentration of 1 ϫ 104 units/ml for 5–10 min led to sufficient induction of tyrosine phosphorylation
Methyl esterification, IMAC, and isotope labeling, we were able to detect tyrosine phosphorylation induced by IFN␣ treatment in Jurkat cells

Summary

Introduction

Tyrosine phosphorylation is a type of post-translational modification that plays a crucial role in signal transduction. Quantitative changes of tyrosine phosphorylation at the global level were evaluated using isotopic labeling (introduced at the methyl esterification step prior to IMAC) Using this double enrichment approach, we characterized interferon ␣ (IFN␣)-induced pTyr proteomic changes in Jurkat cells. Parent ion scanning of phosphate- or pTyr-specific marker fragment ions may be used [4] to detect phosphopeptides in the in-geldigested samples, and phosphorylation sites can be identified by MS/MS These gel-based approaches are not very effective [5] and are not amenable to high throughput studies. Methyl esterification of acidic side chains of peptides has been demonstrated to dramatically reduce the nonspecific background in IMAC-based approaches This strategy has allowed for the identification of over 200 phosphopeptides from a yeast lysate [6]. They are classified as type I IFNs (including IFN␣, IFN␤, and IFN␻) and type II IFN (IFN␥)

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