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United States Department of Agriculture

Agricultural Research Service

Title: Improvements in Robotic Sample Preparation for the Identification of Low Abundance Bacterial Proteins Via Maldi Mass Spectrometry

Author
item Bayles, Darrell

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: March 7, 2005
Publication Date: June 5, 2005
Citation: Bayles, D.O. 2005. Improvements in robotic sample preparation for the identification of low abundance bacterial proteins via maldi mass spectrometry. 105 General Meeting of the American Association for Microbiology. CD ROM ISBN 1-55581-338-0. Paper NO. 0-018.

Technical Abstract: Bacterial proteomics can generate comprehensive catalogs of expressed proteins. Current methods for robotic isolation and purification of proteins from two-dimensional electrophoresis gels (2DE) require high levels of protein comparable to levels that can be detected with Coomassie Blue protein staining methods. Improvements are needed if robotic sample handling of proteins is to lead to effective protein identification using mass spectrometry (MS) of lower abundance proteins detected with sensitive stains like SYPRO Ruby. Using a PerkinElmer MultiProbe II EX liquid handling robot and an Applied Biosystems 4700 Proteomics Analyzer, we have developed improvements in the liquid handling phase of sample preparation that have improved the MS signal-to-noise ratio sufficiently to allow detection and identification of tryptic protein digests at and below one fmole of digested protein. The improvements in robotic sample application feature separate deposition steps for the alpha-cyano-4-hydroxycinnamic acid (CHCA) and peptide samples to a MALDI target stage heated to 50C. Heated dying improved crystallization by favoring production of smaller more homogenous crystals and also minimized sample spreading on stainless-steel MALDI targets. Depositing the CHCA before the peptides increased the signal-to-noise ratio 2 to 3-fold and was dependent upon peptide concentration. Reducing the CHCA concentration to levels between 1.25 and 0.3 mg/ml improved the signal-to-noise ratio up to 5-fold the signal-to-noise observed with 5 mg/ml CHCA. Together these improvements allowed MS identification of proteins at a level more closely matching the amounts of protein that can be detected following SYPRO Ruby staining of 2DE gels and provided sufficient sample to conduct tandem MS of the proteins. Currently the improved method is being used to detect proteins isolated from Listeria monocytogenes.

Last Modified: 9/23/2014
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