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United States Department of Agriculture

Agricultural Research Service

Title: Improved Technique for Robotic Spotting and Detection of Low Abundance Proteins by Maldi-Tof/tof Mass Spectrometry

Authors
item Nunez, Alberto
item Bayles, Darrell
item Fortis, Laurie

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: April 20, 2005
Publication Date: June 5, 2005
Citation: Nunez, A., Bayles, D.O., Fortis, L.L. 2005. Improved technique for robotic spotting and detection of low abundance proteins by maldi-tof/tof mass spectrometry. Meeting Abstract.

Technical Abstract: Proteomics analyses using automated processing of 2D gels and robotic spotting of trypsin digested proteins onto MALDI plates can afford successful identification for most abundant proteins, but low concentration proteins require methods that maximize sensitivity for MALDI/TOF analysis. It is well known that the matrix/analyte ratio plays a significant roll in detection sensitivity, and that the use of high organic solvent concentrations produces the best peptide recovery from C18 cleanup cartridges. However, matrix mixtures with a high content of organic solvent present a number of robotic spotting challenges. In this study, the effect of matrix concentration, spotting sequence (sample over matrix or vise versa), and sample stage heating were evaluated to maximize peptide detection and identification. Trypsin digested peptide mixtures or standard solutions of des-Arg(1)-bradikinin; angiotensin I, and Glu(1) fibrinopeptide B in the concentration range of 1-50 fmols in acetonitrile:H2O (70:30) were spotted onto MALDI plates using CHCA as matrix in the concentration range between 0.3 to 5 mg/ml in acetonitrile:H2O (50:50). The MALDI plate was at room temperature or preheated at 50 degrees C and spotted separately with matrix and peptides as follows: matrix first and then peptides, peptides first and then matrix, manually or using a PerkinElmer MultiProbe II EX robot fitted with a custom plate heater. Mass spectra were obtained with a MALDI-TOF/TOF 4700 Proteomics Analyzer instrument (Applied Biosystems) with database search capabilities via the MASCOT search engine. MALDI-TOF/TOF analysis of peptides of low abundance proteins (amols/*l) that were robotically spotted using methods compatible with automated procedures did not provide sufficient sensitivity for peptide mass fingerprints (PMF), and MS/MS fragmentation analysis was limited. Consequently, the signal to noise ratio (S/N) of the individual ionized peptides was measured following the different spotting approaches described in the Methods section. The results indicate that the best S/N was obtained when the matrix, at a concentration of 0.6 mg/ml, was spotted first followed by spotting the peptides on top of the dried matrix, all at 50 degrees C. Under these conditions, the S/N increased up to 5-fold compared to conventional methods using 5 mg/ml of matrix and spotting at room temperature. To determine the lower limit of detection of peptides and database search confidence, a solution of beta-galactosidase in the concentration range of 25 fmols to 25 amols was evaluated using the optimized procedure. The peptide stability in the mixtures was not affected by heating at 50 degrees C, and afforded improved confirmatory protein scores from database searches even at concentrations as low as 25 amol. This improved procedure is being applied to the analysis of selected proteins extracted using robotic processing of 2D gel samples from Listeria monocytogenes.

Last Modified: 10/21/2014
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