Capillary isoelectric focusing hyphenated to single- and multistage matrix-assisted laser desorption/ionization-mass spectrometry using automated sheath-flow-assisted sample deposition.
In this paper CIEF combined with MALDI-MS is described using a sheath-liquid-assisted automatic sample deposition from the separation capillary onto a MALDI target. Sample/matrix preparation techniques on the target resembling the dried droplet and the thin layer methods were evaluated in the context of the automatic spotting. Volatile buffers were used as IEF catholyte solutions. Test samples consisting of tryptic peptides, glycopeptides, and phosphopeptides of well-known proteins showed that CIEF-MALDI-MS can be used as effective preseparation method prior to MS, allowing to obtain the amino acid sequence coverage of proteins similar to that achieved with CZE-MALDI-MS and CZE-ESI-MS. Particularly, completeness and reliability of glycopeptide analysis is much enhanced by the preseparation. The effect is less pronounced but still significantly found with phosphopeptides present in the test protein. Finally, a test sample of five standard proteins demonstrates the suitability of this technique also for the treatment of intact proteins. This technique has potential to emerge as a faster method analogous and complementary to 2-DE and to IPG-IEF-MALDI-MS demonstrated before by the group of Loo [1].
Lechner
Michael
M
Institute of Analytical Chemistry and Food Chemistry, University of Vienna, Vienna, Austria.
Seifner
Alexandra
A
Rizzi
Andreas M
AM
D000818:Animals; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods; D006020:Glycopeptides / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D017421:Sequence Analysis / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*; D014357:Trypsin
D006020:Glycopeptides; D010446:Peptide Fragments; D010748:Phosphopeptides; D014357:Trypsin