Detection of selenoproteins in human cell extracts by laser ablation-ICP MS after separation by polyacrylamide gel electrophoresis and blotting

Abstract

Laser ablation-ICP MS was optimized for the sensitive detection of selenoproteins in polyacrylamide gel and PVDF membrane after blotting. For this purpose, two interlaboratory reference samples were prepared: glutathione peroxidase band in the gel and on the membrane, respectively. The optimisation was carried out using two systems: 213 nm laser (Newwave)-Agilent 7500ce ICP MS, and a 1030 nm high repetition rate femtosecond laser with galvanometric optics (Novalase)-PE/SCIEX DRCII. Sensitivity and signal-to-noise ratio were benchmarked to those obtained for the same sample by a recently published method in a reference lab. The optimization allowed a 12-fold gain of the S/N ratio during ablation of gels and a 3.5-fold gain in the ablation of blots in comparison with the method using an essentially similar system published by the reference lab. The gain of S/N by increasing ablation surface using the high repetition rate laser was not as spectacular as expected (2.5-fold for the gels and 1.5-fold for the blots) as the background noise increased considerably when a larger surface is ablated due to selenoproteins peak tailing. The study allowed for the first time LA-ICP MS detection of selenoproteins (separated by gel electrophoresis) in human cell extracts with the selenium concentration at the 10 ng ml À1 level.

Privileged Incorporation of Selenium as Selenocysteine in Lactobacillus reuteri Proteins Demonstrated by Selenium-specific Imaging and Proteomics

“…The absolute LOD for Se detection in PAGE gels was 15 pg. The effects of the instrumental parameters and the analytical performance of the method were discussed in detail elsewhere (22,23).…”

Selenoproteome Identification in Inflamed Murine Primary Bone Marrow-Derived Macrophages by Nano-LC Orbitrap Fusion Tribrid Mass Spectrometry

“…In the recent past, semi-quantitative and non-targeted mass spectrometry (MS)-based workflows have proven to be popular without the use of radioactive isotopes. The most widely used techniques to analyze selenoprotein include Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) and laser ablation of isoelectric focused immobilized pH gradient strips coupled to ICP MS detection (IEF-LA-ICP MS) [25][26][27]. Each of these methods have advantages, limitations, and challenges in selenoprotein research (reviewed in detail [28]).…”

Laser ablation ICP-MS for quantitative biomedical applications

“…In most applications reported for heteroatom-protein analysis by PAGE-LA-ICP-MS, the analytes were metal-or semimetal-containing proteins such as selenoproteins [48][49][50][51], as well as phosphoproteins [52][53][54]. In such cases the heteroelements are incorporated into the primary protein structure (pure covalent bonds), so the heteroatom-protein bond is not broken during electrophoretic separation.…”