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

Liang Xue,Pengcheng Wang,Lianshui Wang,Emily Renzi,Predrag Radivojac,Haixu Tang,Randy Arnold,Jian-Kang Zhu,W Andy Tao

doi:10.1074/mcp.o113.027284

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. The label-free LAXIC method was further treated with a linear regression function to accurately measure phosphoproteome responses to osmotic stress in Arabidopsis. Among 2027 unique phosphopeptides identified and 1850 quantified phosphopeptides in Arabidopsis samples, 468 regulated phosphopeptides representing 497 phosphosites have shown significant changes. Several known and novel components in the abiotic stress pathway were identified, illustrating the capability of this method to identify critical signaling events among dynamic and complex phosphorylation. Further assessment of those regulated proteins may help shed light on phosphorylation response to osmotic stress in plants.

Highlights

From the ‡Departments of Biochemistry, Purdue University, West Lafayette, IN 47907; §Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907; ¶School of Informatics and Computing, Indiana University, Bloomington, IN 47405; ʈDepartment of Chemistry, Indiana University, Bloomington, IN 47405;ʈʈShanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China
Design and Characterization of Synthetic Peptide Libraries for Phosphoproteomics—To achieve accurate quantitation using the Library Assisted eXtracted Ion Chromatogram (LAXIC) approach, a well-designed peptide library is required based on the following criteria: [1] Peptides should distribute along the whole chromatogram and cover a wide range of retention time
By combining the self-validating method developed by Casado and Cutillas [47] and the LAXIC approach, we simultaneously evaluated the precision and accuracy of large quantitative phosphoproteomics data generated from complex biological samples

Summary

Introduction

From the ‡Departments of Biochemistry, Purdue University, West Lafayette, IN 47907; §Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907; ¶School of Informatics and Computing, Indiana University, Bloomington, IN 47405; ʈDepartment of Chemistry, Indiana University, Bloomington, IN 47405;ʈʈShanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. 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. For a global quantitative proteomics study, it is unrealistic to spike-in all reference peptides Another labeling reference method, spike-in SILAC appears to be a promising technique to quantify the proteome in vivo with multiplex capability and it can be extended to clinical samples [20]. This quantification occurs in a small time frame centered to each target peptide

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Discussion

Conclusion