The determination of protein phosphorylation on electrophoresis gel blots by laser ablation inductively coupled plasma-mass spectrometry

Abstract

Laser ablation interfaced with inductively coupled plasma-mass spectrometry is described as a new method to determine the presence of phosphorylated proteins on electrophoresis gel blots. The method was applied to the phosphoprotein beta-casein with good detection levels being observed at 16 pmole. Attempts at using the technique to detect beta-casein on electrophoresis gels are also described.

Evaluation of gel electrophoresis conditions for the separation of metal‐tagged proteins with subsequent laser ablation ICP‐MS detection

“…One option is to dry the gel between glass plates for some time, removing the top plate before ablation [16]. …”

“…Separation strategies used are 1-D and 2-D SDS-PAGE under non-reducing and reducing conditions, employing Blue Native and SDS GE in the second dimension [15]. The resulting gels were either ablated directly or electroblotted onto a membrane, which was then ablated [16].…”

Modification of tricine–SDS–PAGE for online and offline analysis of phosphoproteins by ICP-MS

“…The coupling of separation techniques to ICP-MS, such as capillary gel electrophoresis (CGE) [26], GE-LA [27][28][29] and micro liquid chromatography (µLC) [4,[30][31] were important developments in proteomics and genomics studies. The µLC-ICP-MS (pioneering work described by Lehmann and co-workers [32] The recently introduced on-line coupling of GE to ICP-MS is a further development and was first described by Brüchert and Bettmer [35] for the determination of dsDNA fragments.…”

Inductively Coupled Plasma–Mass Spectrometry (ICP-MS) for Quantitative Analysis in Environmental and Life Sciences: A Review of Challenges, Solutions, and Trends

“…The first report with respect to the utilization of LA-ICP-MS for protein phosphorylation analysis was published by Marshall et al, who described the investigation of membrane-blotted phosphorylated proteins by LA-ICP-MS. 172 b-casein was used as model protein and detection limits of 16 pmol were obtained.…”

“…However, due to the high phosphorus background of the gel matrix it was not possible to determine the location of the phosphorylated proteins. 172 To overcome the contamination problem, Wind et al improved this approach by adding a washing step after the blotting process into their strategy for the LA-ICP-MS based analysis of gelseparated phosphoproteins. 173 Ga(NO 3 ) 3 was used as complexing agent to remove any non-covalently bound phosphate from the membrane, which is known to result in non-specific interactions with other non-phosphorylated sample constituents, leading to false positive results.…”