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Realised by ALMS™
developer of the AIDS-HIV Reference project
Abstract No.: ThP-161
Session: Nanotechnology
Presentation date: Thu, Aug 31, 2006
Presentation time: 09:50 – 11:20

Fully Automated Sample Preconcentration and Chip-based Nanoelectrospray

Mark Allen1, Reinaldo Almeida1, Mark Baumert1, Thomas Corso2, Colleen Van Pelt2

1 Advion, Norwich, United Kingdom
2 Advion, Ithaca, United States

Correspondence address: Mark Allen, Advion, Rowan House, 26-28 Queens Road, Norwich, NR3 9DB United Kingdom.

Web site: http://www.advion.com

Keywords: Electrospray Ionization (ESI); Mass Spectrometry; Nanoscale Science/Technology; Protein Identification.

Novel aspect: Fully automated sample concentration and nanoelectrospray.

 

Introduction
Nanoelectrospray and NanoLC are the preferred techniques for analysis where sample size is limited. Developed by Wilm & Mann in 1996 nanoelectrospray show a high detection efficiency by the fact that the droplets generated are already small enough to produce gas-phase ions directly (Karas, Bahr, & Dulcks, 2000).A few nl/min flow rates assume that very low amount of sample can be used for long analyses time. However it is difficult to automate and handle this low amounts and therefore the sample is diluted typically into 2-5 無. Here we describe a fully automated pre concentration and nanoelectrospray analysis using
the TriVersa Nanomate (Advion BioSciences), a chip-based electrospray system, designed to achieve low volume and low flow-rate sample introduction.

Methods
Samples were desalted and eluted into a 96 well plate with 5 無 75:25 methanol/water 0.1 % FA. ESI-MS analyses were carried out by direct infusion mass spectrometry using the TriVersa Nanomate system. The robot was programmed to aspirate 2 無 into a conductive pipette tip and then deliver to the electrospray chip at a flow rate of around 20nL/min. In a second experiment a concentration step was added to the infusion cycle, by programming the robot to aspirate 2 無 sample into a conductive tip and partially dispense it, so that a droplet is exposed to the ambient allowing a fast evaporation.The sample evaporated down to around 200 nL within 1 min and then analyzed automatically.

Preliminary results
The sample pre concentration step of 2 無 to 200 nL show a big sensitivity enhancement compared to the direct infusion. It was possible to spray the sample for 10 minutes giving a flow rate of around 20nL/min. This was enough time to fully characterize the sample with good MS/MS data. The concentration step could easily be modified in order to have longer spray time if needed. Different test compounds were analysed and their ESI response investigated before and after the concentration step. In addition, the lowest concentration at which MS/MS data could be generated was determined. In this way, it was possible to have better detection limits with ESI-MS without adding any complexity to the analyses or compromising analysis time. Finally, in order to determine the relevance of the approach, tryptic digests were analysed and the detection limit investigated by identifying the protein with a database search. The conclusion of these studies is that the high sensitivity of direct infusion mass spectrometry with a preconcentration step facilitate the rapid Identification of Proteins separated by gel electrophoreses and therefore increases the sample throughput.