Abstract
Phosphopeptide enrichment is a commonly used sample preparation step for investigating phosphorylation. TiO2-based enrichment has been demonstrated to have excellent performance both for large amounts of complex and for small amounts of simple samples. However, it has not yet been studied for complex samples in the nanogram range. Our objective was to develop a methodology applicable for complex samples in the low nanogram range, useful for mass spectrometry analysis of tissue microarrays. The selectivity and performance of two stationary phases (TiO2 nanoparticle-coated monolithic column and spin tip filled with TiO2 microspheres) and several loading solvents were studied. Based on this study, we developed an effective and robust method, based on a spin tip with a non-conventional 50 mM citric acid-based loading solvent. It gave excellent results for phosphopeptide enrichment from samples containing a few nanograms of a complex protein mixture.
Highlights
Phosphorylation is one of the most widely investigated post-translational modifications (PTMs) of proteins
Optimize, and describe the performance of various TiO2 -metal oxide affinity chromatography (MOAC) systems for the enrichment of small amounts of complex samples carrying out a detailed performance test of two different stationary phases and various sample loading solvent combinations
We have thoroughly analyzed the performance of two stationary phases and multiple loading solvents for phosphopeptide enrichment originating from very small amounts of complex samples
Summary
Phosphorylation is one of the most widely investigated post-translational modifications (PTMs) of proteins. This ubiquitous PTM is responsible for several biological functions—for example, signal transduction, switching on/off the functionality of a protein, or growth regulation during cancer [1]. Mass spectrometry (MS) is an established and powerful analytical technique with its ever-growing sensitivity, low-abundance PTMs are still overlooked during the analysis due to sensitivity limitations. Taking all these into consideration, it is advisable to perform an enrichment/isolation step before MS measurements. The most common way is to Separations 2020, 7, 74; doi:10.3390/separations7040074 www.mdpi.com/journal/separations
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