CE coupled to MALDI with novel covalently coated capillaries.
CE offers the advantage of flexibility and method development options. It excels in the area of separation of ions, chiral, polar and biological compounds (especially proteins and peptides). Masking the active sites on the inner surface of a bare fused silica capillary wall is often necessary for CE separations of basic compounds, proteins and peptides. The use of capillary surface coating is one of the approaches to prevent the adsorption phenomena and improve the repeatability of migration times and peak areas of these analytes. In this study, new capillary coatings consisting of (i) derivatized polystyrene nanoparticles and (ii) derivatized fullerenes were investigated for the analysis of peptides and protein digest by CE. The coated capillaries showed excellent run-to-run and batch-to-batch reproducibility (RSD of migration time < or = 0.5% for run-to-run and < or = 9.5% for batch-to-batch experiments). Furthermore, the capillaries offer high stability from pH 2.0 to 10.0. The actual potential of the coated capillaries was tested by combining CE with MALDI-MS for analysing complex samples, such as peptides, whereas the overall performance of the CE-MALDI-MS system was investigated by analysing a five-protein digest mixture. Subsequently, the peak list (peptide mass fingerprint) generated from the mass spectra of each fraction was entered into the Swiss-Prot database in order to search for matching tryptic fragments using the MASCOT software. The sequence coverage of analysed proteins was between 36 and 68%. The established technology benefits from the synergism of high separation efficiency and the structure selective identification via MS.
Bachmann
Stefan
S
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens, University Innsbruck, Austria.
Vallant
Rainer
R
Bakry
Rania
R
Huck
Christian W
CW
Corradini
Danilo
D
Bonn
Günther K
GK
D030562:Databases, Protein; D053841:Electroosmosis; D019075:Electrophoresis, Capillary / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D037741:Fullerenes / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007840:Latex / Q000737:chemistry*; D053758:Nanoparticles / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D011137:Polystyrenes / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D037741:Fullerenes; D007840:Latex; D010446:Peptide Fragments; D011137:Polystyrenes; D011506:Proteins; D014357:Trypsin