Quantitative Measurement of Phosphoproteome Response to Osmotic Stress in Arabidopsis Based on Library-Assisted eXtracted Ion Chromatogram (LAXIC)

Abstract

Global phosphorylation changes in plants in response to environmental stress have been relatively poorly characterized to date. Here we introduce a novel mass spectrometry-based label-free quantitation method that facilitates systematic profiling plant phosphoproteome changes with high efficiency and accuracy. This method employs synthetic peptide libraries tailored specifically as internal standards for complex phosphopeptide samples and accordingly, a local normalization algorithm, LAXIC, which calculates phosphopeptide abundance normalized locally with co-eluting library peptides. Normalization was achieved in a small time frame centered to each phosphopeptide to compensate for the diverse ion suppression effect across retention time. Phosphorylation plays a pivotal role in the regulation of a majority of cellular processes via signaling transduction pathways. During the last decade, quantitative phosphoproteomics has become a powerful and versatile platform to profile signaling pathways at a system-wide scale. Multiple signaling networks in different organisms have been characterized through global phosphorylation profiling (1-3), which has evolved over the years with highly optimized procedures for sample preparation and phosphopeptide enrichment, high resolution mass spectrometry, and data analysis algorithms to identify and quantify thousands of phosphorylation events (4 -8).Quantitative phosphoproteomics can be achieved mainly by two major techniques, stable isotope labeling and labelfree quantitation. Isotope labeling prior to liquid chromatography-mass spectrometry (LC-MS) 1 has been widely used, including metabolic labeling such as stable isotope labeling by amino acids in cell culture (SILAC), chemical labeling such as multiplexed isobaric tags for relative and absolute quantification (iTRAQ) and isotope-coded affinity tags (ICAT) (9 -12). On the other hand, label-free quantitation has gained momentum in recent years (13-15), as no additional chemistry or sample preparation steps are required. Compared with stable isotope labeling, label-free quantitation has higher compatibility with the source of the samples, the number of samples for comparison, and the instrument choice.Many label-free approaches, in particular to phosphoproteomics, are based on ion intensity (16, 17), but they are relatively error-prone because of run-to-run variations in LC/MS performance (18). In theory, such systematic errors can be corrected by spiking an internal standard into every sample to be compared. Several methods based on internal standard spiking have been reported so far. Absolute quantification peptide technology (AQUA) (19), for example, uses synthetic peptides with isotope labeling for absolute quanti- 1 The abbreviations used are: LC-MS, liquid chromatography-MS; ABA, abscisic acid; CV, coefficient of variation; FDR, false discovery rate; GO, gene ontology; LAXIC, Library Assisted eXtracted Ion Chromatogram; PBS, phosphate buffered saline; PolyMAC, polymerbased metal-ion affinity capture; SnRK, SNF-related ser...

Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action

“…This suggests that along with SnRK2.2, 2.3, and 2.6, ABA also may activate other protein kinases to phosphorylate these proteins. Previous studies have shown that SnRK2.4, 2.7, and 2.8 could be weakly activated by ABA (7,14). However, genetic evidence demonstrated that in planta ABA sensitivity depends almost entirely on SnRK2.2/2.3/2.6, and that SnRK2.4/2.7/2.8 have little influence (8,9).…”

“…The SEQUEST database search algorithm was used for peptide sequence identification and determination of modification sites. Ion intensity-based label-free quantitation (14) in the parent ion mode was performed to quantify the amount of phosphopeptides across four samples. Using this strategy, a total of 5,386 unique phosphopeptides (5,084 phosphosites) belonging to 2,243 unique phosphoproteins were identified with a 1% false discovery rate (Dataset S1).…”

“…In recent years, mass spectrometry-based global phosphorylation profiling has evolved to where it is able to dissect thousands of phosphorylation events simultaneously in vivo (14). To comprehensively examine the regulation by phosphorylation in response to ABA and the role of the SnRK2s, we quantitatively compared the phosphoproteomes of WT and snrk2.2/2.3/2.6 triple mutant under ABA treatment.…”

Phosphoproteomic Analyses Reveal Early Signaling Events in the Osmotic Stress Response

“…2B). bZIP30 phosphorylation has been shown to be responsive to exogenous ABA treatment to a lesser extent (Kline et al, 2010;Xue et al, 2013) and to 30 min of mannitol treatment (Xue et al, 2013), but an earlier osmotic challenge has not been examined previously. Within the bZIP class I subfamily, two proteins closely related to bZIP29 and bZIP30 have been reported to play a role in the abiotic stress response (Fig.…”

Regulation of Leaf Starch Degradation by Abscisic Acid Is Important for Osmotic Stress Tolerance in Plants

“…Conversely, there is no evidence for redox regulation of BAM3, and the enzyme has optimum activity at less alkaline pH (Santelia et al, 2015;Monroe et al, 2014). Furthermore, large-scale site-specific phosphorylation profiling of Arabidopsis proteins revealed the presence of one or more phosphorylation sites on both BAM1 and AMY3 proteins (de la Fuente van Bentem et al, 2008;Reiland et al, 2009;Xue et al, 2013;Heazlewood et al, 2008). Given the rapid activation of leaf starch degradation in response to the osmotic stress, it is likely that posttranslational modifications such as protein phosphorylation or redox regulation played a major role in activating BAM1 and AMY3 in the light.…”