Systematic studies on TiO2-based phosphopeptide enrichment procedures upon in-solution and in-gel digestions of proteins. Are there readily applicable protocols suitable for matrix-assisted laser desorption/ionization mass spectrometrybased phosphopeptide stability estimations?

作者：Eickner Thomas; Mikkat Stefan; Lorenz Peter; Sklorz Martin; Zimmermann Ralf; Thiesen Hans Juergen; Glocker Michael O^*

来源：European Journal of Mass Spectrometry, 2011, 17(5): 507-523.

DOI：10.1255/ejms.1134

• TiO2
• titanium dioxide
• phosphorylation
• phosphopeptide enrichment
• dephosphorytation
• citric acid
• NuTips
• phosphopeptide analysis
• MALDI-MS
• mass spectrometry
• affinity mass spectrometry
• chemical dephosphorylation

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 TiO2-loaded pipette tips in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analyses. Using beta-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 (DNB) 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.

• 出版日期2011