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A simple and reliable protocol for mouse serum proteome profiling studies by use of two-dimensional electrophoresis and MALDI TOF/TOF mass spectrometry

: Ritorto, M.S.; Borlak, J.

Volltext urn:nbn:de:0011-n-855154 (1.2 MByte PDF)
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Erstellt am: 15.1.2009

Proteome science. Online journal (2008), Nr.6, Art. 25
ISSN: 1477-5956
Zeitschriftenaufsatz, Elektronische Publikation
Fraunhofer ITEM ()
serum proteome; proteomic - method; genetic technique; mass spectrometry

Background: Unravelling the serum proteome is the subject of intensified research. In this regard, two-dimensional electrophoresis coupled with MALDI MS analysis is still one of the most commonly used method. Despite some improvements, there is the need for better protocols to enable comprehensive identification of serum proteins. Here we report a combination of two proteomic strategies, zoom in acidic and neutral part of 2-D gels and an application of two optimised matrix preparations for MALDI-MS analyses to simplify serum proteome mapping. Results: Mouse serum proteins were separated by 2-D electrophoresis at the pH ranges 3-10 and 4-7, respectively. Then in gel tryptic digests were analysed by MALDI-MS. Notably, sample-matrix preparations consisted of either a thin-layer alpha-ciano-4-hydroxycinnamic acid (CHCA) matrix deposition or a matrix-layer 2,5-dihydroxybenzoic acid (DHB). This enabled an identification of 90 proteins. The herein reported method enhanced identification of proteins by 32% when compared with previously published studies of mouse serum proteins, using the same approaches. Furthermore, experimental improvements of matrix preparations enabled automatic identification of mouse proteins, even when one of the two matrices failed. Conclusion: We report a simple and reliable protocol for serum proteome analysis that combines an optimized resolution of 2-D gels spots and improved sample-matrix preparations for MALDI-MS analysis. The protocol allowed automated data acquisition for both CHCA and DHB and simplified the MS data acquisition therefore avoiding time-consuming procedures. The simplicity and reliability of the developed protocol may be applied universally.