Enrichment strategies for phosphoproteomics: state-of-the-art

Abstract

Protein phosphorylation is a key regulator in many biological processes, such as homeostasis, cellular signaling and communication, transcriptional and translational regulation, and apoptosis. The defects in this tightly controlled reversible posttranslational modifi cation have been described to contribute to genesis and progression of various diseases, emphasizing the importance of a systematic research of this phenomenon. Although considerable effort has been devoted to improving the analysis of phosphorylation by mass spectrometry, which is currently the method of choice to study protein phosphorylation, the detection and identifi cation of phosphorylation sites remains challenging because of the low abundance and low ionization effi cacy of phosphoproteins in comparison with nonphosphorylated proteins. To overcome this obstacle, different enrichment strategies for phosphorylated peptides/proteins have been established and optimized for subsequent mass spectrometry analysis. In this review, we will give an overview of the methods currently available for the enrichment of phosphorylated proteins and peptides including immunoprecipitation, chemical derivatization and affi nity enrichment techniques.

Guanidinium functionalized superparamagnetic silica spheres for selective enrichment of phosphopeptides and intact phosphoproteins from complex mixtures

“…Systematic identication and characterization of phosphoproteins is vital to help us understand this important modication; however, it remains a challenging task due to the relatively low abundance of phosphorylated proteins in biological samples. [3][4][5] Current approaches to capture phosphoproteins generally rely on the interaction of phosphate anions with metal ions, such as in immobilized metal affinity chromatography (IMAC) and in metal oxide affinity chromatography (MOAC). [6][7][8][9][10] Even though these techniques have been demonstrated with success to a certain extent, some disadvantages are encountered with these techniques: including poor selectivity, low adsorption efficiency and signicant loss of phosphorylated proteins during the wash steps.…”

Zinc Ion-immobilized Magnetic Microspheres for Enrichment and Identification of Multi-phosphorylated Peptides by Mass Spectrometry

“…Thus, the study of protein phosphorylation states is important for defining protein kinase substrates, and revealing the activation states of signal transduction pathways. [5][6][7] Currently, high-throughput proteomic analysis is mainly accomplished by mass spectrometry (MS). 8 However, the determination of phosphopeptides in complex biological samples has remained a significant challenge because their scarcity in the total proteome as well as their deficient ionization and ion suppression during the mass spectrometric analysis.…”

ZrO2 Nanofiber as a Versatile Tool for Protein Analysis

“…Substantial research efforts have been devoted to improve separation and identification of phosphoproteome by chromatographic techniques and mass spectrometry. − Still, this remains a challenging task, because phosphopeptides are present at very low abundancetypically <5% in a complex protein digestand their MS signals could be suppressed by the nonphosphorylated ones. One effective solution is to selectively enrich the phosphorylated proteins or peptides prior to MS detection. , …”

“…One effective solution is to selectively enrich the phosphorylated proteins or peptides prior to MS detection. 9,10 Antibodies or phosphoprotein-binding domains (PBDs) have been employed to isolate phosphorylated proteins or peptides with high specificity. 11,12 However, neither is readily available and both are expensive to obtain.…”