Synthesis of Magnetic Microspheres with Immobilized Metal Ions for Enrichment and Direct Determination of Phosphopeptides by Matrix‐Assisted Laser Desorption Ionization Mass Spectrometry

By Xiuqing Xu, Chunhui Deng, Mingxia Gao, Wenjia Yu, Pengyuan Yang, Xiangmin Zhang. 2006

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Phosphorylation, one of the most common and important post-translational modifications of proteins, plays a critical role in signal-transduction, regulatory, and metabolic pathways. [1] With the motivation of understanding these biopro-cesses, much effort has been devoted to the development of methods to identify the specific sites of phosphorylation. Recently, mass-spectrometric techniques based on MALDI-TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) has emerged as a powerful tool for phosphorylation site mapping. However, the signals of the phos-phopeptides are often suppressed by nonphosphorylated-pep-tide residues contained in protein digests, which means that the identification of phosphorylated peptides is still challenging. Therefore, separation and enrichment of phosphorylated peptides from proteolytic digest mixtures is in urgent demand. To meet this requirement, immobilized metal ion affinity chromatography (IMAC) [2] has been employed to selectively purify phosphorylated peptides, prior to MS analysis. By use of this method, acidic phosphopeptides retained on porous resin beads can be directly characterized by MALDI-TOF MS, which allows sequencing of the enriched phosphopeptides without the necessity for prior elution of the peptides from the beads, thus avoiding sample loss during the elution process. Unfortunately, due to the “shadow effect” [3] of porous resin beads, a large number of phosphopeptides bound in the pores are not fully accessible during direct laser desorption. Zhang et al. [4] recently reported the enrichment of phosphorylated

Subfields: Molecular Biology, Spectroscopy

View Paper: https://www.semanticscholar.org/paper/0ffa76c430b81692d2d6be7ac5b100f13c17d540

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