Nanoflow liquid chromatography coupled to matrix-assisted laser desorption/ionization mass spectrometry: sample preparation, data analysis, and application to the analysis of complex peptide mixtures.
We report the development of a robust interface for off-line coupling of nano liquid chromatography (LC) to matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) and its application to the analysis of proteolytic digests of proteins, both isolated and in mixtures. The interface makes use of prestructured MALDI sample supports to concentrate the effluent to a small sample plate area and localize the MALDI sample to a predefined array, thereby enriching the analyte molecules and facilitating automated MALDI-MS analysis. Parameters that influence the preparation of MALDI samples from the LC effluent were evaluated with regard to detection sensitivity, spectra quality, and reproducibility of the method. A procedure for data processing is described. The presented nano LC MALDI-MS system allowed the detection of several peptides from a tryptic digest of bovine serum albumin, at analyzed amounts corresponding to one femtomole of the digested protein. For the identification of native proteins isolated from mouse brain by two-dimensional gel electrophoresis, nano LC MALDI-MS increased the number of detected peptides, thereby allowing identification of proteins that could not be identified by direct MALDI-MS analysis. The ability to identify proteins in complex mixtures was evaluated for the analysis of Escherichia coli 50S ribosomal subunit. Out of the 33 expected proteins, 30 were identified by MALDI tandem time of flight fragment ion fingerprinting.
Mirgorodskaya
Ekaterina
E
Max Planck Institute for Molecular Genetics, Berlin, Germany. migorod@molgen.mpg.de
Braeuer
Corina
C
Fucini
Paola
P
Lehrach
Hans
H
Gobom
Johan
J
D000818:Animals; D001923:Brain Chemistry; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D029968:Escherichia coli Proteins / Q000032:analysis; D013058:Mass Spectrometry; D051379:Mice; D036103:Nanotechnology; D010449:Peptide Mapping; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D015203:Reproducibility of Results; D012269:Ribosomal Proteins / Q000032:analysis / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods; D014357:Trypsin / Q000378:metabolism
D029968:Escherichia coli Proteins; D010455:Peptides; D012269:Ribosomal Proteins; D012710:Serum Albumin, Bovine; D014357:Trypsin