Spotting and quantification of phosphoproteins purified by gel electrophoresis and laser ablation‐element mass spectrometry with phosphorus‐31 detection

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Abstract
Laser ablation inductively coupled plasma-mass spectrometry (ICP-MS) with 31P detection has been used for spotting of phosphoproteins after one-dimensional polyacrylamide gel electrophoresis (1-D PAGE) and membrane transfer. By analyzing a mixture of myoglobin, α-casein and reduced fibrinogen it is demonstrated that phosphoproteins are specifically recognized by this method. A special washing step was found to be necessary to remove phosphate noncovalently bound to proteins. The 31P signal was found to contain quantitative information both with respect to relative and absolute amounts of phosphorus present in phosphoproteins. Normalizing the 31P signal from a single laser ablation trace by the total amount of phosphoprotein applied to the gel, a detection limit of 5 pmol of phosphorus is estimated.

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