17th International Mass Spectrometry Conference :: Prague, 2006
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|Session:||Proteomics: New Methods|
|Presentation date:||Tue, Aug 29, 2006|
|Presentation time:||09:50 – 11:20|
Juan Astorga-Wells1,2, Susanne Vollmer1, Tomas Bergman1, Hans Jornvall1 Karolinska Institutet, Stockholm, Sweden
Correspondence address: Juan Astorga-Wells, Karolinska Institutet, Medical Biochemistry and Biophysics, Scheeles vag 2, Stockholm, SE-171 77 Stockholm Sweden.
Keywords: MALDI; Microanalysis; Proteomic; Separations.
Novel aspect: Novel separation method (and a multifunctional tool) specially suitable for proteome discovery by mass spectrometry.
Miniaturization is an important requirement in the design of analytical devices for high-throughput applications. In this respect, the combination of microfluidic electrocapture before mass spectrometry has emerged as an analytical alternative to micro-LC-MS in the analysis of biomolecules. The electrocapture technology allows immobilization/separation of charged molecules in a microflow stream by the counteracting effects of hydrodynamic and electric forces. Using electrocapture, desalting, concentration, buffer exchange, removal of contaminants, online multistep microreactions and peptide separation can all be obtained. In this presentation we describe the use of this multifunctional tool for the separation of peptides and proteins with special emphasis on the separation before mass spectrometry.
The device consists of a capillary tubing with two small openings covered with a tubular conductive membrane. The conductive junctions are placed in separate electrode chambers, by which an electric field can be generated inside the channel. Upstream, the device is connected to an injector and a syringe pump. Downstream, the device is either left open for subsequent spotting onto the target for MALDI-MS or online connected to ESI-MS. The syringe pump and power supply are controlled and monitored using a software developed in-house. The software allows programming of different gradient protocols with voltage steps as low as 10 mV/min.
Successful combination of electrocapture-based separations and MS was achieved using solutions in low mM concentration of TrisHCl. A MALDI Q-Tof mass spectrometer was used for the peptide sequence analysis. Tryptic peptides from digestions of mixtures of proteins were separated by electrocapture. Compared to the unseparated sample, better protein sequence coverage was achieved. The resolution of the separation was inversely proportional to the slope of the voltage gradient, with gradient times in the order of 1.5 h. The technique proved to be particularly suitable for MALDI spotting since it works with flow rates similar to those of micro-LC (200 nl/min). Also, the fact that the gradient is made by the voltage -and not by the solvent composition- makes the system less prone to failure. As seen from the physical characteristics of the identified peptides, the instrument separates peptides according to charge without use of salt gradients. This makes electrocapture-based separations suitable as an alternative to or a replacement of ion-exchange chromatography and for online connection to reverse-phase chromatography for two-dimensional separations. We continue these applications focusing on the analysis of cell extracts, and in parallel also conduct similar studies to find the best approach to combine electrocapture-based separations with ESI-MS/MS analysis.