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




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Proteome Science

, 6:25

First Online: 12 September 2008Received: 21 May 2008Accepted: 12 September 2008

Abstract

BackgroundUnravelling 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.

ResultsMouse 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 α-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.

ConclusionWe 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.

List of abbreviationsACNacetonitril

DTTdithiothreitol

IPGimmobilized pH gradient

MALDI-TOF-MSMatrix Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry

NH4HCO3ammonium bicarbonate

SDSSodium Dodecyl Sulfate

TCAtrichloroacetic acid

TFAtrifluoracetic acid

TEMEDtetramethylenediamine.

Electronic supplementary materialThe online version of this article doi:10.1186-1477-5956-6-25 contains supplementary material, which is available to authorized users.

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Autor: Maria Stella Ritorto - Jürgen Borlak

Fuente: https://link.springer.com/







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