Performance of Isobaric and Isotopic Labeling in Quantitative Plant Proteomics

Nogueira, Fábio César Sousa; Palmisano, Giuseppe; Schwämmle, Veit; Campos, Francisco; Larsen, Martin R.; Domont, Gilberto B.; Roepstorff, Peter

doi:10.1021/pr300192f

Cited by 43 publications

(33 citation statements)

References 26 publications

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“…The percentage labeling efficiency was calculated based on the total number of high confidence unique peptide sequence identifications (denominator, determined at a 1% false discovery rate (FDR) cutoff using Percolator) and the number of unique peptides carrying a TMT label (numerator) which was considered as variable during the database search 5 (see Table 1). Irrespective of the instrument used and the resulting analytical depth, the labeling efficiencies of the two protocols, i.e.…”

Section: Resultsmentioning

confidence: 99%

iFASP: Combining Isobaric Mass Tagging with Filter-Aided Sample Preparation

2013

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Careful, clean and controlled preparation of samples for mass spectrometry proteomics is crucial to obtain reproducible and reliable data. This is especially important when carrying out quantitative proteomics by chemical isobaric labeling (aka tandem mass tagging) since the differentially labeled samples are combined quite late during the sample processing. Addressing this need for robust and reliable sample processing for quantitative proteomics, we describe here iFASP, a simple protocol for combining isobaric mass tagging with the recently introduced Filter-Aided Sample Preparation (FASP) method. iFASP provides a quick, simple and effective method for obtaining clean samples, ensuring efficient digestion and providing excellent labeling yields for quantitative proteomics experiments. We have carried out our iFASP protocol using several highly complex Xenopus laevis egg and embryo lysates and compared the labeling yields and number of high-confidence peptide identifications to a standard in-solution digestion and labeling protocol. Although the labeling efficiency with both techniques is in the 99+% range, the number of peptides identified with a 1% false discovery rate and the corresponding number of quantified peptide spectral matches are as much as doubled with iFASP compared to the corresponding non-FASP-based method.

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Section: Resultsmentioning

confidence: 99%

iFASP: Combining Isobaric Mass Tagging with Filter-Aided Sample Preparation

2013

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“…From the ratios of the ion intensities of these sister peptide pairs, the relative abundance of their parent proteins in the original samples can be determined [68]. Recently, a detailed experimental protocol called postdigest ICPL was published highlighting a better protein identification and quantification [69, 70] and when compared to iTRAQ, both techniques have shown comparable number of identified and quantified proteins in the endosperm of castor bean seeds at three developmental stages [71]. So far, the latter study is the unique reported quantitative proteomic investigation on plants using ICPL (Table 2).…”

Section: Overview Of Label-based Proteomic Approachesmentioning

confidence: 99%

Gel-Based and Gel-Free Quantitative Proteomics Approaches at a Glance

Abdallah

Dumas‐Gaudot

Renaut³

et al. 2012

International Journal of Plant Genomics

168

175

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Two-dimensional gel electrophoresis (2-DE) is widely applied and remains the method of choice in proteomics; however, pervasive 2-DE-related concerns undermine its prospects as a dominant separation technique in proteome research. Consequently, the state-of-the-art shotgun techniques are slowly taking over and utilising the rapid expansion and advancement of mass spectrometry (MS) to provide a new toolbox of gel-free quantitative techniques. When coupled to MS, the shotgun proteomic pipeline can fuel new routes in sensitive and high-throughput profiling of proteins, leading to a high accuracy in quantification. Although label-based approaches, either chemical or metabolic, gained popularity in quantitative proteomics because of the multiplexing capacity, these approaches are not without drawbacks. The burgeoning label-free methods are tag independent and suitable for all kinds of samples. The challenges in quantitative proteomics are more prominent in plants due to difficulties in protein extraction, some protein abundance in green tissue, and the absence of well-annotated and completed genome sequences. The goal of this perspective assay is to present the balance between the strengths and weaknesses of the available gel-based and -free methods and their application to plants. The latest trends in peptide fractionation amenable to MS analysis are as well discussed.

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“…In contrast to Arabidopsis, castor bean (Ricinus communis), a member of Euphorbiaceae family, has relatively large and persistent endosperm throughout seed development (Greenwood and Bewley, 1982). It has been considered as a model plant for studies on endosperm and seed development among dicots (Houston et al, 2009;Nogueira et al, 2012). Also, castor bean is one of the most important nonedible oilseed crops, and its seed oil stored in endosperm is used broadly in industry (Ogunniyi, 2006).…”

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confidence: 99%

Genomic DNA methylation analyses reveal the distinct profiles in castor bean seeds with persistent endosperms

Yang

Dong

et al. 2016

Plant Physiol.

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Investigations of genomic DNA methylation in seeds have been restricted to a few model plants. The endosperm genomic DNA hypomethylation has been identified in angiosperm, but it is difficult to dissect the mechanism of how this hypomethylation is established and maintained because endosperm is ephemeral and disappears with seed development in most dicots. Castor bean (Ricinus communis), unlike Arabidopsis (Arabidopsis thaliana), endosperm is persistent throughout seed development, providing an excellent model in which to dissect the mechanism of endosperm genomic hypomethylation in dicots. We characterized the DNA methylation-related genes encoding DNA methyltransferases and demethylases and analyzed their expression profiles in different tissues. We examined genomic methylation including CG, CHG, and CHH contexts in endosperm and embryo tissues using bisulfite sequencing and revealed that the CHH methylation extent in endosperm and embryo was, unexpectedly, substantially higher than in previously studied plants, irrespective of the CHH percentage in their genomes. In particular, we found that the endosperm exhibited a global reduction in CG and CHG methylation extents relative to the embryo, markedly switching global gene expression. However, CHH methylation occurring in endosperm did not exhibit a significant reduction. Combining with the expression of 24-nucleotide small interfering RNAs (siRNAs) mapped within transposable element (TE) regions and genes involved in the RNA-directed DNA methylation pathway, we demonstrate that the 24-nucleotide siRNAs played a critical role in maintaining CHH methylation and repressing the activation of TEs in persistent endosperm development. This study discovered a novel genomic DNA methylation pattern and proposes the potential mechanism occurring in dicot seeds with persistent endosperm.

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Performance of Isobaric and Isotopic Labeling in Quantitative Plant Proteomics

Cited by 43 publications

References 26 publications

iFASP: Combining Isobaric Mass Tagging with Filter-Aided Sample Preparation

iFASP: Combining Isobaric Mass Tagging with Filter-Aided Sample Preparation

Gel-Based and Gel-Free Quantitative Proteomics Approaches at a Glance

Genomic DNA methylation analyses reveal the distinct profiles in castor bean seeds with persistent endosperms

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