Abstract

Protein post-translational modifications (PTMs) are among the fastest and earliest of plant responses to changes in the environment, making the mechanisms and dynamics of PTMs an important area of plant science. One of the most studied PTMs is protein phosphorylation. This review summarizes the use of targeted proteomics for the elucidation of the biological functioning of plant PTMs, and focuses primarily on phosphorylation. Since phosphorylated peptides have a low abundance, usually complex enrichment protocols are required for their research. Initial identification is usually performed with discovery phosphoproteomics, using high sensitivity mass spectrometers, where as many phosphopeptides are measured as possible. Once a PTM site is identified, biological characterization can be addressed with targeted proteomics. In targeted proteomics, Selected/Multiple Reaction Monitoring (S/MRM) is traditionally coupled to simple, standard protein digestion protocols, often omitting the enrichment step, and relying on triple-quadruple mass spectrometer. The use of synthetic peptides as internal standards allows accurate identification, avoiding cross-reactivity typical for some antibody based approaches. Importantly, internal standards allow absolute peptide quantitation, reported down to 0.1 femtomoles, also useful for determination of phospho-site occupancy. S/MRM is advantageous in situations where monitoring and diagnostics of peptide PTM status is needed for many samples, as it has faster sample processing times, higher throughput than other approaches, and excellent quantitation and reproducibility. Furthermore, the number of publicly available data-bases with plant PTM discovery data is growing, facilitating selection of modified peptides and design of targeted proteomics workflows. Recent instrument developments result in faster scanning times, inclusion of ion-trap instruments leading to parallel reaction monitoring- which further facilitates S/MRM experimental design. Finally, recent combination of data independent and data dependent spectra acquisition means that in addition to anticipated targeted data, spectra can now be queried for unanticipated information. The potential for future applications in plant biology is outlined.

Highlights

  • Protein post-translational modifications (PTMs) are among the fastest and earliest of plant responses to changes in the environment, making the mechanisms and dynamics of PTMs an important area of plant science

  • This review summarizes the use of targeted proteomics for the elucidation of the biological functioning of plant PTMs, and focuses primarily on phosphorylation

  • Recent instrument developments result in faster scanning times, inclusion of ion-trap instruments leading to parallel reaction monitoring- which further facilitates Selected/Multiple Reaction Monitoring (S/MRM) experimental design

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Summary

SRM for Plant PTM

PHOSPHORYLATION: IMPORTANCE IN REGULATION OF PLANT PROCESSES AND MOST COMMON TECHNIQUES FOR MASS-SPECTROMETRIC ANALYSIS. As a product of enzymatic activity, it was first discovered at the beginning of the twentieth century (Levene and Alsberg, 1906; Burnett and Kennedy, 1954) It is controlled by a fine balance between kinases and phosphatases (Schulze, 2010). The critical step in MS analysis involves detection of the loss of phosphate (neutral loss 98 kDa) from serine (Ser), threonine (Thr), and tyrosine (Tyr) in MS3. This occurs after the peptide has been fragmented to its amino acids. After the initial SRM assay is developed, the speed and throughput for processing samples significantly increases

OVERVIEW OF TARGETED PROTEOMICS AND CONSIDERATIONS FOR STUDY OF PHOSPHOPEPTIDES
DESIGN OF SRM EXPERIMENTS FOR TARGETING PROTEIN PHOSPHORYLATION
EXAMPLES OF TARGETED PROTEOMICS IN THE ANALYSIS OF PHOSPHORYLATED PEPTIDES
Medicago Arabidopsis
Findings
CONCLUSIONS AND VIEW AHEAD
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