Improved Electrophoretic Separation to Assist the Monitoring of Bcl-xL Post-Translational Modifications

Abstract

Bcl-xL is an oncogene of which the survival functions are finely tuned by post-translational modifications (PTM). Within the Bcl-2 family of proteins, Bcl-xL shows unique eligibility to deamidation, a time-related spontaneous reaction. Deamidation is still a largely overlooked PTM due to a lack of easy techniques to monitor Asn→Asp/IsoAsp conversions or Glu→Gln conversions. Being able to detect PTMs is essential to achieve a comprehensive description of all the regulatory mechanisms and functions a protein can carry out. Here, we report a gel composition improving the electrophoretic separation of deamidated forms of Bcl-xL generated either by mutagenesis or by alkaline treatment. Importantly, this new gel formulation proved efficient to provide the long-sought evidence that even doubly-deamidated Bcl-xL remains eligible for regulation by phosphorylation.

1D continuous gel electrophoresis composition for the separation of deamidated proteins

“…A total amount of 250 ng of protein was incubated at 37 • C in various deamidation solutions (final volume 250 µL) supplemented with 10 mM DTT to prevent dimerization. Untreated WT recombinant Bcl-xL was used as the non-deamidated control, while recombinant Bcl-xL N52D/N66D was used as a migration standard for double deamidation as in [23]. 10 ng of proteins were separated on 15% polyacrylamide gels.…”

“…In the original mini-gel composition we described in [23], chloride composes the leading phase of high mobility ions while Taurine and Glycinate compose the trailing phase of low mobility ions. The stacking/ destacking of proteins to be separated is ensured by the shift of pH between the stacking gel (pH = 6.7) and the resolving gel (pH = 8.8), and the change of the pore size from 5% in the stacking gel to 12% or 15% in the resolving gel.…”

“…We initially designed Taurine/Glycinate gels to assist the separation of Asn deamidated proteins [23]. We set out to determine whether these gels could also be used to separate Gln deamidated proteins.…”

“…The use of Taurine/Glycinate as trailing ions allowed us to pioneer the description of the sequential deamidation of the cellular oncogene Bcl-xL [23]. This 26 kDa protein adopts the classical fold characteristic of Bcl-2 family members, namely a globular protein organized in alpha helices.…”

“…S2 in [22]). But these methods rely on multi-step analyses and to overcome this drawback, we recently described a modified electrophoretic method using Taurine/Glycinate as trailing ions to allow, on mini-gels, a fast and accurate detection of the sequential deamidation of Asn52 and Asn66 in Bcl-xL [23].…”