Multiple marker abundance profiling: combining selected reaction monitoring and data‐dependent acquisition for rapid estimation of organelle abundance in subcellular samples

Hooper, Cornelia M.; Stevens, Tim J.; Saukkonen, Anna Maija; Castleden, Ian; Singh, Pragya; Mann, Gregory W.; Fabre, Bertrand; Ito, Jun; Deery, Michael J.; Lilley, Kathryn S.; Kim, Young-Mo; Millar, A. Harvey; Heazlewood, Joshua L.; Parsons, Harriet T.

doi:10.1111/tpj.13743

Cited by 13 publications

(13 citation statements)

References 54 publications

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“…This is mostly because the fractionation of mitochondria from Arabidopsis seeds is practically impossible and thus the identification and quantitation of mitochondrial proteins must rely on the use of the whole seed protein extract. With the rapid advancement of mass spectrometry techniques, the application of targeted proteomics for the detection and quantitation of mitochondrial proteins in complex samples obtained from plant material has become a promising analytical tool in such studies (Hooper et al, 2017 ). In this work, we presented MRM as a sensitive and specific technique for estimating mitochondrial protein abundances in dry and germinating Arabidopsis seeds without the organelle enrichment.…”

Section: Discussionmentioning

confidence: 99%

“…The recent development of a mass spectrometry-based targeted proteomics approach, such as Multiple Reaction Monitoring (MRM: the acquisition of multiple product ions from one precursor ion by Selected Reaction Monitoring (SRM) transitions), and its utilization for the quantitative analysis of peptides derived from proteins of specific organelles in plant cells has become a powerful, hypothesis-driven analytical tool to achieve specific, sensitive, and precise protein quantification (Zulak et al, 2009 ; Hall et al, 2011 ; Fan et al, 2012 ; Hooper et al, 2017 ). The high specificity of MRM analysis comes from the mass measurement (accurately measured to an atomic mass unit) of the whole peptide and its multiple fragments, the unique peptide ion fragmentation pattern, and the precise peptide retention time in the liquid chromatography (LC) separation (Liebler and Zimmerman, 2013 ).…”

Section: Introductionmentioning

confidence: 99%

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Targeted Proteomics Approach Toward Understanding the Role of the Mitochondrial Protease FTSH4 in the Biogenesis of OXPHOS During Arabidopsis Seed Germination

et al. 2018

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Seed germination provides an excellent model to study the process of mitochondrial biogenesis. It is a complex and strictly regulated process which requires a proper biogenesis of fully active organelles from existing promitochondrial structures. We have previously reported that the lack of the inner mitochondrial membrane protease FTSH4 delayed Arabidopsis seed germination. Here, we implemented a targeted mass spectrometry-based approach, Multiple Reaction Monitoring (MRM), with stable-isotope-labeled standard peptides for increased sensitivity, to quantify mitochondrial proteins in dry and germinating wild-type and ftsh4 mutant seeds, lacking the FTSH4 protease. Using total seed protein extracts we measured the abundance of the peptide targets belonging to the OXPHOS complexes, AOX1A, transport, and inner membrane scaffold as well as mitochondrial proteins that are highly specific to dry and germinating seeds. The MRM assay showed that the abundance of these proteins in ftsh4 did not differ substantially from that observed in wild-type at the level of dry seed and after stratification, but we observed a reduction in protein abundance in most of the examined OXPHOS subunits in the later stages of germination. These changes in OXPHOS protein levels in ftsh4 mutants were accompanied by a lower cytochrome pathway activity as well as an increased AOX1A amount at the transcript and protein level and alternative pathway activity. The analyses of the steady-state transcript levels of mitochondrial and nuclear genes encoding OXPHOS subunits did not show significant difference in their amount, indicating that the observed changes in the OXPHOS occurred at the post-transcriptional level. At the time when ftsh4 seeds were fully germinated, the abundance of the OXPHOS proteins in the mutant was either slightly lowered or comparable to these amounts in wild-type seeds at the similar developmental stage. By the implementation of an integrative approach combining targeted proteomics, quantitative transcriptomics, and physiological studies we have shown that the FTSH4 protease has an important role in the biogenesis of OXPHOS and thus biogenesis of mitochondria during germination of Arabidopsis seeds.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Targeted Proteomics Approach Toward Understanding the Role of the Mitochondrial Protease FTSH4 in the Biogenesis of OXPHOS During Arabidopsis Seed Germination

et al. 2018

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“…LOPIT resulted in the identification of 345 ER-, 46 TGN-, and 397 Golgi-resident proteins in three spatially distinct clusters, along with comprehensive lists of resident protein markers for all other organelles (Supplemental Figure 1; Supplemental Data Set 1). The currently annotated Arabidopsis Golgi proteome (covering all cell types) is estimated at ;530 proteins (Hooper et al, 2017b), suggesting that we identified a large majority of resident Golgi proteins present in our cell line.…”

Section: Establishing Updated Subproteomes For the Golgi And Other Ormentioning

confidence: 91%

Separating Golgi Proteins from Cis to Trans Reveals Underlying Properties of Cisternal Localization

et al. 2019

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The order of enzymatic activity across Golgi cisternae is essential for complex molecule biosynthesis. However, an inability to separate Golgi cisternae has meant that the cisternal distribution of most resident proteins, and their underlying localization mechanisms, are unknown. Here, we exploit differences in surface charge of intact cisternae to perform separation of early to late Golgi subcompartments. We determine protein and glycan abundance profiles across the Golgi; over 390 resident proteins are identified, including 136 new additions, with over 180 cisternal assignments. These assignments provide a means to better understand the functional roles of Golgi proteins and how they operate sequentially. Protein and glycan distributions are validated in vivo using high-resolution microscopy. Results reveal distinct functional compartmentalization among resident Golgi proteins. Analysis of transmembrane proteins shows several sequence-based characteristics relating to pI, hydrophobicity, Ser abundance, and Phe bilayer asymmetry that change across the Golgi. Overall, our results suggest that a continuum of transmembrane features, rather than discrete rules, guide proteins to earlier or later locations within the Golgi stack.

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“…To find targeted processes or protein hotspots sensitive to Ssulfenylation, we first determined the subcellular distribution of the identified S-sulfenylated proteins ( Fig. 2A and Dataset S1) (28,29). The best-represented subcellular compartments are cytoplasm (48%), chloroplast (18%), mitochondrion (13%), and nucleus (12%).…”

Section: Bioinformatics Analysis On the Target Specificity Of S-sulfementioning

confidence: 99%

Mining for protein S-sulfenylation in Arabidopsis uncovers redox-sensitive sites

Huang

Willems

Wei

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

121

115

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Hydrogen peroxide (H2O2) is an important messenger molecule for diverse cellular processes. H2O2 oxidizes proteinaceous cysteinyl thiols to sulfenic acid, also known as S-sulfenylation, thereby affecting the protein conformation and functionality. Although many proteins have been identified as S-sulfenylation targets in plants, site-specific mapping and quantification remain largely unexplored. By means of a peptide-centric chemoproteomics approach, we mapped 1,537 S-sulfenylated sites on more than 1,000 proteins in Arabidopsis thaliana cells. Proteins involved in RNA homeostasis and metabolism were identified as hotspots for S-sulfenylation. Moreover, S-sulfenylation frequently occurred on cysteines located at catalytic sites of enzymes or on cysteines involved in metal binding, hinting at a direct mode of action for redox regulation. Comparison of human and Arabidopsis S-sulfenylation datasets provided 155 conserved S-sulfenylated cysteines, including Cys181 of the Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE4 (AtMAPK4) that corresponds to Cys161 in the human MAPK1, which has been identified previously as being S-sulfenylated. We show that, by replacing Cys181 of recombinant AtMAPK4 by a redox-insensitive serine residue, the kinase activity decreased, indicating the importance of this noncatalytic cysteine for the kinase mechanism. Altogether, we quantitatively mapped the S-sulfenylated cysteines in Arabidopsis cells under H2O2 stress and thereby generated a comprehensive view on the S-sulfenylation landscape that will facilitate downstream plant redox studies.

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Multiple marker abundance profiling: combining selected reaction monitoring and data‐dependent acquisition for rapid estimation of organelle abundance in subcellular samples

Cited by 13 publications

References 54 publications

Targeted Proteomics Approach Toward Understanding the Role of the Mitochondrial Protease FTSH4 in the Biogenesis of OXPHOS During Arabidopsis Seed Germination

Targeted Proteomics Approach Toward Understanding the Role of the Mitochondrial Protease FTSH4 in the Biogenesis of OXPHOS During Arabidopsis Seed Germination

Separating Golgi Proteins from Cis to Trans Reveals Underlying Properties of Cisternal Localization

Mining for protein S-sulfenylation in Arabidopsis uncovers redox-sensitive sites

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