Rapid identification of phosphorus containing proteins in electrophoresis gel spots by Laser-Induced Breakdown Spectroscopy, LIBS
A novel method for the rapid in-gel identification of phosphorus containing proteins, specifically casein and ovalbumin, prior to mass spectrometric analysis for the elucidation of phosphorylation sites was developed. After polyacrylamide gel-electrophoretic separation, staining and drying, protein bands were subjected to focused laser pulses at the center or the vicinity of the protein band. Phosphorus containing proteins were recognized from their prominent phosphorus lines in the luminous plasma formed by energetic laser pulses. The LIBS emission intensity of phosphorus lines at 253.5 nm and 255.3 nm has been optimized with respect to laser energy and detector timing parameters by using pure casein in the pellet form. The method was applied to casein, ovalbumin, two commercially available standard protein mixtures and proteins extracted from the canola plant. It was shown that LIBS was capable of identifying phosphorus containing proteins directly in the gel matrix in nanogram amounts. Mass spectrometric analysis of the ovalbumin spot after the in-gel digestion procedure has proved the accuracy of the technique. With the speed and spatial resolution that LIBS offers, this technique shows promise in the micro-local spotting of phosphorus containing proteins in the polyacrylamide gel matrix prior to MS analysis for the determination of the phosphorylation sites.