Rapid and reproducible phosphopeptide enrichment by tandem metal oxide affinity chromatography: application to boron deficiency induced phosphoproteomics

Abstract

Mass spectrometry has been instrumental in enabling the study of molecular signaling on a cellular scale by way of site-specific quantification of protein post-translational modifications, in particular phosphorylation. Here we describe an updated tandem metal oxide affinity chromatography (MOAC) combined phosphoprotein/phosphopeptide enrichment strategy, a scalable phosphoproteomics approach that allows rapid identification of thousands of phosphopeptides in plant materials. We implemented modifications to several steps of the original tandem MOAC procedure to increase the amount of quantified phosphopeptides and hence site-specific phosphorylation of proteins in a sample beginning with the less amounts of tissue and a substantially smaller amount of extracted protein. We applied this technology to generate time-resolved maps of boron signaling in Arabidopsis roots. We show that the successive enrichment of phosphoproteins in a first and phosphopeptide extraction in a second step using our optimized procedure strongly enriched the root phosphoproteome. Our results reveal that boron deficiency affects over 20% of the measured root phosphoproteome and that many phosphorylation sites with known biological function, and an even larger number of previously undescribed sites, are modified during the time course of boron deficiency. We identify transcription factors as key regulators of hormone signaling pathways that modulate gene expression in boron deprived plants. Furthermore, our phosphorylation kinetics data demonstrate that mitogen-activated protein kinase (MAPK) cascades mediate polarized transport of boron in Arabidopsis roots. Taken together, we establish and validate a robust approach for proteome-wide phosphorylation analysis in plant biology research.

Exploring the diversity of plant proteome

“…In addition, multidimensional fractionation of protein samples is useful for enhancing the depth of phosphoproteome coverage, but this technique usually requires large amounts of starting material and long measurement times. Recent developed methods, such as tandem MOAC and EasyPhos, capture unexpected signaling targets and are powerful techniques for achieving in‐depth phosphoproteome coverage within hours and with fewer sample preparation steps (Hoehenwarter et al, 2013; Humphrey et al, 2018; Chen and Hoehenwarter, 2019).…”

“…One example is the phosphorylation of activation loops in a kinase domain, resulting in the regulation of a downstream pathway. Thus, quantitative phosphoproteomics has been used for proteome‐wide mapping of the substrates of particular protein kinases, such as the sucrose nonfermenting‐related kinase (SnRK), mitogen‐activated protein (MAP) kinase, calcium‐dependent protein kinase (CDPK), or receptor‐like kinases (Cox and Mann, 2012; Hoehenwarter et al, 2013; Umezawa et al, 2013; Wang et al, 2013; Chen and Hoehenwarter, 2019; Wu et al, 2019).…”

Labeling and label-free quantitative proteomics in plant biology

“…)信息 [49] . 在典型的串联质谱实验中, 一 级离子通常要被进一步打碎成片段(MS 的磷酸化水平被不同程度调控, 例如硼转运体蛋白 (BOR2和BOR3) [12] . 后期的功能研究发现, 硼信号通过 激活植物MPK激酶信号通路, 从而磷酸化BOR2和 BOR3, 激活其转运活性来调控物质的运输 [12] .…”

In situ synthesis of a novel metal oxide affinity chromatography affinity probe for the selective enrichment of low‐abundance phosphopeptides

“…12 Among the enrichment strategies that have been developed so far, 13,14 metal oxide affinity chromatography (MOAC), 15,16 which is based on reversible Lewis acid-base interactions between the metal oxides and the negatively charged phosphate groups to achieve the specific selection of phosphopeptides, is one of the most widely used and the one with the most potential. 17,18 Various metal oxides, such as lanthanum oxide, 19 zirconium oxide, 20 and titania, 21 have been developed and widely used for the effective enrichment of phosphopeptides. [19][20][21] Further research on MOAC has led to metal oxides being modified on various substrates, such as diatomite, magnetic Fe 3 O 4 , 22 and graphene (G), to enrich phosphopeptides.…”

“…17,18 Various metal oxides, such as lanthanum oxide, 19 zirconium oxide, 20 and titania, 21 have been developed and widely used for the effective enrichment of phosphopeptides. [19][20][21] Further research on MOAC has led to metal oxides being modified on various substrates, such as diatomite, magnetic Fe 3 O 4 , 22 and graphene (G), to enrich phosphopeptides. 23 Among these substrates, carbon-based materials have become a significant group due to their good physical and chemical properties.…”