Spotting and quantification of phosphoproteins purified by gel electrophoresis and laser ablation-element mass spectrometry with phosphorus-31 detection.
Laser ablation inductively coupled plasma-mass spectrometry (ICP-MS) with (31)P 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, alpha-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 (31)P signal was found to contain quantitative information both with respect to relative and absolute amounts of phosphorus present in phosphoproteins. Normalizing the (31)P 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.
Wind
Mathias
M
Central Spectroscopy, German Cancer Research Center (DKFZ), Heidelberg, Germany.
Feldmann
Ingo
I
Jakubowski
Norbert
N
Lehmann
Wolf D
WD
D015153:Blotting, Western; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D007834:Lasers; D013058:Mass Spectrometry / Q000379:methods*; D010750:Phosphoproteins / Q000032:analysis / Q000302:isolation & purification*; D010759:Phosphorus Isotopes / Q000032:analysis*
D010750:Phosphoproteins; D010759:Phosphorus Isotopes; D017868:Cyclic AMP-Dependent Protein Kinases