Global Analysis of Gel Mobility of Proteins and Its Use in Target Identification

Atsuko Shirai,Akihisa Matsuyama,Yoko Yashiroda,Atsushi Hashimoto,Yumi Kawamura,Ritsuko Arai,Yasuhiko Komatsu,Sueharu Horinouchi,Minoru Yoshida

doi:10.1074/jbc.m709211200

Abstract

SDS-PAGE is a basic method that has long been used for separation of proteins according to their molecular sizes. Despite its simplicity, it provides information on characteristics of proteins beyond their molecular masses because gel mobility of proteins often reflects their physicochemical properties and post-translational modifications. Here we report on a global analysis of gel mobility of the proteome, which we term the "mobilitome," covering 93.4% of the fission yeast proteome. To our surprise, more than 40% of proteins did not migrate to their calculated positions. Statistical analyses revealed that the discrepancy was largely dependent on the hydrophobicity of proteins. This experimental data set, with a high coverage rate of real mobility, made it feasible to identify proteins detected on the gel without using any specialized techniques. This approach enabled us to detect previously unknown post-translational modifications of a protein; for example, we revealed that eIF5A is novel substrate of a Sir2-related deacetylase Hst2. Furthermore, we concomitantly identified twelve acetylated and eight methylated proteins using specific anti-acetylated and anti-methylated lysine antibodies, most of which had not been known to be subject to the modifications. Thus, we propose the general usefulness of the mobilitome and electrophoresis-based methodology for the identification and characterization of proteins detected on the gel.

Highlights

We identified several novel acetylated or methylated proteins, using a global screen based on induced expression of the tagged proteins in combination with Western blotting (WB)
The Mobilitome, an “-omic” Data Set Based on Gel Mobility of Proteins—To set up a strategy for global analysis of gel mobility of proteins in SDS-PAGE, we constructed S. pombe integrants in which each of the ORFs fused with two copies of the FLAG epitope and six consecutive histidine residues tag (His6) at the 3Ј terminus is integrated at the chromosomal leu1 locus [13]
This strain collection was established based on a Gateway-cloned ORFeome resource that covered more than 99% of genes predicted by the genome project [7]

Summary

Introduction

The Mobilitome, an “-omic” Data Set Based on Gel Mobility of Proteins—To set up a strategy for global analysis of gel mobility of proteins in SDS-PAGE, we constructed S. pombe integrants in which each of the ORFs fused with two copies of the FLAG epitope and six consecutive histidine residues tag (His6) at the 3Ј terminus is integrated at the chromosomal leu1 locus [13] (supplemental Fig. S1A). The overall size distribution of proteins measured by gel mobility was not significantly different from that of proteins calculated from their amino acid sequences (p ϭ 0.060, as determined by the Mann-Whitney U test) (supplemental Fig. S2).

Results

Conclusion