Document Detail

EJMS Protocol: Systematic studies on TiO(2)-based phosphopeptide enrichment procedures upon in-solution and in-gel digestions of proteins. Are there readily applicable protocols suitable for MALDI-MS-based phosphopeptide stability estimations?
MedLine Citation:
PMID:  22173543     Owner:  NLM     Status:  In-Data-Review    
There have been many successful efforts to enrich phosphopeptides in complex protein mixtures by the use of immobilized metal affinity chromatography (IMAC) and/or metal oxide affinity chromatography (MOAC) with which mass spectrometric analysis of phosphopeptides has become state of the art in specialized laboratories, mostly applying nanoLC electrospray ionization mass spectrometry-based investigations. However, widespread use of these powerful techniques is still not achieved. In this study, we present a ready-to-use phosphopeptide enrichment procedure using commercially available TiO(2)-loaded pipette tips in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analyses. Using α-casein as a model protein and citric acid as additive during sample loading, a similar enrichment success can be achieved as compared to applying 2,5- dihydroxy benzoic acid (DHB) for this task. But the DHB-inherited drawbacks are eliminated. In addition, we show that combining DHB and 2,4,6-trihydroxy acetophenone (THAP) as matrix for MALDI-MS measurements retains the sensitivity of DHB for phosphopeptide analysis but adds the homogenous crystallization properties of THAP, enabling preparation of evenly distributed matrix surfaces on MALDI-MS anchor targets, a prerequisite for automated MALDI- MS analyses. Tripartite motif-containing protein 28 and stathmin are two examples for which successful phosphopeptide enrichment of either sodium dodecyl sulfate polyacrylamide gel electrophoresis or two-dimensional gel electrophoresis-separated proteins is shown. Finally, high resolution MALDI Fourier transform ion cyclotron resonance mass spectrometry after phosphopeptide enrichment suggests that chemical dephosphorylation may occur as a side reaction during basic elution of phosphopeptides bound to MOAC surfaces, suggesting that proteome-wide phosphopeptide analyses ought to be interpreted with caution. In contrast, in-depth analysis of phosphopeptide/non-phosphorylated peptide siblings may be used to estimate stability differences of phosphorylation sites in individual proteins, possibly adding valuable information on biological regulation processes.
Thomas Eickner; Stefan Mikkat; Peter Lorenz; Martin Sklorz; Ralf Zimmermann; Hans-Jürgen Thiesen; Michael O Glocker
Related Documents :
22216433 - Multi-walled carbon nanotube composites with polyacrylate prepared for open-tubular cap...
22209303 - Design and evaluation of hydrolytically stable bidentate urea-type stationary phases fo...
18210383 - Identification and quantification of methylglyoxal as the dominant antibacterial consti...
Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  European journal of mass spectrometry (Chichester, England)     Volume:  17     ISSN:  1469-0667     ISO Abbreviation:  Eur J Mass Spectrom (Chichester, Eng)     Publication Date:  2011  
Date Detail:
Created Date:  2011-12-16     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101124748     Medline TA:  Eur J Mass Spectrom (Chichester, Eng)     Country:  England    
Other Details:
Languages:  eng     Pagination:  507-23     Citation Subset:  IM    
Proteome Center Rostock, Medical Faculty, University of Rostock, Rostock, Germany.
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Previous Document:  Differential interactions of isomeric amino sugars with insulin studied under electrospray ionisatio...
Next Document:  Hypertension, cardiovascular risk and polymorphisms in genes controlling the cytochrome P450 pathway...