Silicone/graphite coating for on-target desalting and improved peptide mapping performance of matrix-assisted laser desorption/ionization-mass spectrometry targets in proteomic experiments.
In two-dimensional gel electrophoresis-based proteomic experiments matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) peptide mass fingerprinting is often the technique of choice in identifying proteins. Here, we present a novel surface coating technique for MALDI-MS targets that improves manual and automatic sample analysis. A mixture of silicone and graphite is spread in the form of a thin layer over the target. Due to the hydrophobicity of the coating, aqueous solutions can be applied to relatively small spots very precisely using a robotic system. At least four times more liquid can be concentrated on the same area compared to uncoated steel targets. alpha-cyano-4-hydrocinnamic acid crystallizes in form of very small crystals evenly distributed over the surface. The search for "hot spots" during the analysis is not necessary, which supports the automatic acquisition of data. The homogeneous crystal layer can be very effectively washed on-target without encountering major sample losses. This efficient washing and the focused application of aqueous samples replace expensive and time-consuming reversed phase micro column based sample clean-ups. When analyzing peptide mixtures, the signal intensities are up to five times higher than with preparations of the same un-desalted samples on steel targets, since four times more sample can be loaded. The mass resolution remains unaffected by the surface coating. After usage the coating can be removed, followed by a new coating avoiding any carry-over of sample to the next analysis. All these properties make the precoating of MALDI-MS targets with a silicone/graphite layer an ideal technique for routine analysis in large-scale proteomic experiments.
Li
Xinping
X
European Molecular Biology Laboratory, Proteomic Core Facility, Heidelberg, Germany.
Wilm
Matthias
M
Franz
Thomas
T
D001322:Autoanalysis; D003373:Coumaric Acids / Q000737:chemistry; D003460:Crystallization; D016208:Databases, Factual; D029968:Escherichia coli Proteins / Q000032:analysis; D006108:Graphite; D057927:Hydrophobic and Hydrophilic Interactions; D010449:Peptide Mapping; D010455:Peptides / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D012492:Salts; D012828:Silicones; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013232:Steel / Q000737:chemistry; D013499:Surface Properties
D003373:Coumaric Acids; D029968:Escherichia coli Proteins; D010455:Peptides; D012492:Salts; D012828:Silicones; D013232:Steel; C007175:alpha-cyano-4-hydroxycinnamate; D006108:Graphite