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Improved method for identification of low abundance proteins using 2D-gel electrophoresis, MALDI-TOF and TOF/TOF
Citation:
WINNIK, W. M., O. Alzate, M. E. BRUNO, L. BURGOON, Y. GE, G. R. KLINEFELTER, PRASADA RAO S. KODAVANTI, J. B. Robinette, J. D. SUAREZ, AND K. WALLACE. Improved method for identification of low abundance proteins using 2D-gel electrophoresis, MALDI-TOF and TOF/TOF. Presented at 58th American Society for Mass Spectrometry Conference, Salt Lake City, UT, May 22 - 27, 2010.
Impact/Purpose:
This abstract describes a new analytical approach created by Winnik and applied to 3 projects managed by the Pis: Vue Ge (IRP# NHEERL -ECD-MTB-YG-2007-02; Gary Klinefelter MYP: EDC-1; Prasada Kodavanti LTG: HH-1: Common mode of action, APM (10), MYP: HHR
Description:
Introduction: Differential protein expression studies have been routinely performed in our laboratory to determine the health effects of environmentally-important chemicals. In this abstract, improvements in the in-gel protein digestion, MALDI plate spotting and data acquisition are described. This technique allows protein identification from challenging samples: two year old gels or fresh mini gels loaded with low amounts of protein. The procedure eliminates tube-to-tube sample transfer and hence sample loss by taking the full advantage of ZipTip protein digest extraction and plating. Multiple spots are made per-sample to increase the number of MSMS spectra and protein coverage. The first round of protein identification is performed using MALDIMS data and auto-acquired MALDI-MSMS spectra, followed by the targeted MALDI-MSMS to maximize protein coverage. Method: Protein digestion: 2D-gel spots are picked either manually or automatically by a spot picker. The automatically picked mini-gel plugs are easily lost due to electrostatic charging unless all liquid handling (syringe) and gel plug drying (SpeedVac) are performed through small apertures made with a needle in the Eppendorf tube caps. The gel plugs are destained twice in an ammonium bicarbonate solution, dehydrated by acetonitrile and dried in a SpeedVac. A small volume of trypsin solution is added, the tubes placed in a Thermomixer (300rpm, 3JOC), secured with aluminum foil and incubated for 3 hours. Additional 20-30ul of a digestion solution is added and the incubation continues overnight. The digestion is terminated with TFA and peptide extraction continues for 1h. Preliminary data: Multiple rat testes proteins, differentially-expressed after a phthalate treatment of the animals, were identified from a mini-gel (30ug loaded on a gel, auto-picked plugs; Krypton gel stain). Multiple differentially-expressed arsenic-induced human keratinocyte proteins were identified in Sypro Ruby-stained gels after two-year storage at 4°C, and differentially-expressed mouse brain proteins were identified from a 2D gel stained with Sypro Ruby. Plating: the peptides are extracted directly from the solution above the gel plugs using C18 ZipTips, desalted and eluted as four spots onto a MALDI plate in a minimal volume of CHCA MALDI matrix solution in 80% acetonitrile-20% water. After the extraction, each peptide-depleted solution is returned back to its original tube and incubated for 1h (300rpm, 3JOC) to give a second batch of peptide extract. MALDI analysis: A MALDI-MS peak list from a gel blank digest is added to a MSMS precursor exclusion list of the ABI 4800 instrument control software. Each MALDI spot provides MSMS spectra for 6 MS precursors: spot#1: top intensity precursors 1-6, spot#2: 7-12, spot#3: 13-18. Data analysis: The MSMS data is processed using the Protein Pilot 3.0 software (ABI, Inc). Chemical background-subtracted MALDI-MS data are processed on-line using the Aldente software and the combined reports are used to identify any additional lower-intensity MS peptide peaks missed as precursors in the automated MSMS acquisition. A targeted MALDIMSMS analysis is then performed using the fourth MALDI spot and/or an additional spot that can be plated for each sample using the second-round of the gel plug peptide extraction. The optimized sample preparation, data acquisition and processing allows efficient protein identification from old gel spots, gels stained by various dyes and mini gels loaded with a low protein amount. Novel aspect: In-one-tube: gel destaining, digestion, extraction. High-coverage old gel and minigel spot ID. Protein screening, then targeted MALDI-MSMS for improved ID. Disclaimer: This abstract does not necessarily reflect EPA policy.