Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Reproducibility:  Implication of Multiple Injections

Chong, Poh Kuan; Gan, Chee Lip; Pham, Trong Khoa; Wright, Phillip C.

doi:10.1021/pr060018u

Cited by 182 publications

(235 citation statements)

References 37 publications

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“…For experiment 3, protein extraction, digestion and labelling was performed once. Mass spectrometry analysis of each four-plex sample was performed twice to increase protein coverage and confidence (Chong et al, 2006). Data from all mass spectrometry analyses within each replicate were searched together in ProteinPilot 1.0 (Applied Biosystems, Foster City, CA, USA).…”

Section: Experimental Designmentioning

confidence: 99%

Proteomic characterization of a temperature-sensitive conditional lethal in Drosophila melanogaster

et al. 2009

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Genetic variation that is expressed only under specific environmental conditions can contribute to additional adverse effects of inbreeding if environmental conditions change. We present a proteomic characterization of a conditional lethal found in an inbred line of Drosophila melanogaster. The lethal effect is apparent as a large increase in early mortality at the restrictive temperature (29 1C) as opposed to normal survival at the permissive temperature (20 1C). The increased mortality in response to the restrictive temperature is probably caused by a single recessive major locus. A quantitative trait locus (QTL) region segregating variation affecting the lethal effect has been identified, allowing for a separation of primary/causal effects and secondary consequences in the proteome expression patterns observed. In this study, the proteomic response to the restrictive temperature in the lethal-line (L-line) was compared with the response in an inbred-control-line (IC-line) and an outbred-control-line (OC-line). Quantitative protein changes were detected using isobaric tags for relative and absolute quantitation (iTRAQ) two-dimensional liquid chromatography-tandem mass spectrometry. In all, 45 proteins were found to be significantly differently regulated in response to the restrictive temperature in the L-line as compared with the IC-line. No proteins were significantly differently regulated between the IC-line and the OC-line, verifying that differential protein regulation was specific to a genetic defect in the L-line. Proteins associated with oxidative phosphorylation and mitochondria were significantly overrepresented within the list of differentially expressed proteins. Proteins related to muscle contraction were also found to be differentially expressed in the L-line in response to the restrictive temperature, supporting phenotypic observations of moribund muscle hypercontraction.

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Section: Experimental Designmentioning

confidence: 99%

Proteomic characterization of a temperature-sensitive conditional lethal in Drosophila melanogaster

et al. 2009

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“…Recently, iTRAQ has been adopted for high-efficacy and high-throughput proteomic analysis of a variety of biological samples [34][35][36][37][38][39][40][41][42][43] such as cancer tissues, cells, plants, microorganisms, and sperm. In our study, we combined an easily accessible extraction buffer and a heat-induced antigen retrieval technique to retrieve the proteins from spermatozoa-like cells derived from human iPS transfected with miR-122 or mutant miR-122, followed by high-throughput proteomic iTRAQ analysis to identify and quantify proteins that are differentially expressed between the two groups.…”

Section: Discussionmentioning

confidence: 99%

“…Cell protein extraction, digestion, and labeling with iTRAQ reagents and data analysis All steps of protein extraction and labeling were according to the previously described [34][35][36][37][38][39][40][41][42][43]. Protein identification and quantification for iTRAQ samples were carried out using ProteinPilot software (version 3.0; Applied Biosystems, MDS-Sciex).…”

Section: Teratoma Formationmentioning

confidence: 99%

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MicroRNA-122 Influences the Development of Sperm Abnormalities from Human Induced Pluripotent Stem Cells by Regulating TNP2 Expression

Liu

Huang²,

Liu

et al. 2013

Stem Cells and Development

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Sperm abnormalities are one of the main factors responsible for male infertility; however, their pathogenesis remains unclear. The role of microRNAs in the development of sperm abnormalities in infertile men has not yet been investigated. Here, we used human induced pluripotent stem cells to investigate the influence of miR-122 expression on the differentiation of these cells into spermatozoa-like cells in vitro. After induction, mutant miR-122-transfected cells formed spermatozoa-like cells. Flow cytometry of DNA content revealed a significant increase in the haploid cell population in spermatozoa-like cells derived from mutant miR-122-transfected cells as compared to those derived from miR-122-transfected cells. During induction, TNP2 and protamine mRNA and protein levels were significantly higher in mutant miR-122-transfected cells than in miR-122-transfected cells. High-throughput isobaric tags for relative and absolute quantification were used to identify and quantify the different protein expression levels in miR-122-and mutant miR-122-transfected cells. Among all the proteins analyzed, the expression of lipoproteins, for example, APOB and APOA1, showed the most significant difference between the two groups. This study illustrates that miR-122 expression is associated with abnormal sperm development. MiR-122 may influence spermatozoa-like cells by suppressing TNP2 expression and inhibiting the expression of proteins associated with sperm development.

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“…The first two applications of iTRAQ in cyanobacteria, by Chong et al and Gan et al, published in 2006 and 2007 by our laboratory were fairly successful [52,53]. Both works presented a combined iTRAQ analysis of the cyanobacterium Synechocystis PCC 6803, the crenarchaeal strain Sulfolobus solfataricus P2, and S. cerevisiae to describe the reproducibility and to quantify the biological variation of iTRAQ measurements.…”

Section: Assessment Of the Reproducibility Of Itraq In Cyanobacteria mentioning

confidence: 97%

Current trends in high throughput proteomics in cyanobacteria

Wright

2009

FEBS Letters

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a b s t r a c tAdvancements in genome sequencing and high throughput proteomics of cyanobacterial strains led to 13 published reports, from a small number of laboratories. These successful studies focused on Synechocystis, Nostoc and Anabaena strains, prochlorococcus, and halotolerant Euhalothece. The implications of emerging quantitative aspects developed and applied in these large-scale studies are assessed in the wake of advanced cyanobacterial research. Furthermore, contributions from traditional and early high throughput analysis of cyanobacterial proteomics are compared and summarised. Finally, opinions are provided to link both the trends and the future challenges. This review aims to push the synergy between proteomics and cyanobacterial research to improve both the technical and biological significance.

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Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Reproducibility: Implication of Multiple Injections

Cited by 182 publications

References 37 publications

Proteomic characterization of a temperature-sensitive conditional lethal in Drosophila melanogaster

Proteomic characterization of a temperature-sensitive conditional lethal in Drosophila melanogaster

MicroRNA-122 Influences the Development of Sperm Abnormalities from Human Induced Pluripotent Stem Cells by Regulating TNP2 Expression

Current trends in high throughput proteomics in cyanobacteria

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