Response of peptide intensity to concentration in ESI-MS-based proteome

Lu, Wenyuan; X, Yin; Liu, Xiaohui; Yan, Guoquan; Yang, Pengyuan

doi:10.1007/s11426-014-5096-9

Cited by 3 publications

(5 citation statements)

References 36 publications

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“…The final dehydration step was performed using 100 µL acetonitrile. Trypsinization (1.5 µg trypsin) was performed at 37 • C for 16 h. The digested peptides were centrifuged at 6000× g for 10 min to collect the supernatant and stored at −20 • C (protocol modified from [17]. The samples were lyophilized and 30 µL of the dissolution buffer (0.5 M TEAB, pH 8.5) was added to each sample.…”

Section: Tube-gel Digestion and Sample Clean Upmentioning

confidence: 99%

“…The overwhelming occurrence of vitellogenin yolk proteins is a limiting factor in a polylecithal embryo, such as in zebrafish, as it hinders global identification of less abundant proteins using mass spectrometry-based techniques [4,16]. Proteolytic peptides of yolk proteins can potentially subdue the ionization of the less abundant proteolytic peptides of non-yolk proteins [17,18]. Consequently, abundant yolk proteins can potentially interfere with the identification of cellular proteins, although the degree of such interference is unknown.…”

Section: Introductionmentioning

confidence: 99%

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Proteomics Analysis of Early Developmental Stages of Zebrafish Embryos

Purushothaman

Das

Presslauer

et al. 2019

IJMS

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Zebrafish is a well-recognized organism for investigating vertebrate development and human diseases. However, the data on zebrafish proteome are scarce, particularly during embryogenesis. This is mostly due to the overwhelming abundance of egg yolk proteins, which tend to mask the detectable presence of less abundant proteins. We developed an efficient procedure to reduce the amount of yolk in zebrafish early embryos to improve the Liquid chromatography–tandem mass spectrometry (LC–MS)-based shotgun proteomics analysis. We demonstrated that the deyolking procedure resulted in a greater number of proteins being identified. This protocol resulted in approximately 2-fold increase in the number of proteins identified in deyolked samples at cleavage stages, and the number of identified proteins increased greatly by 3–4 times compared to non-deyolked samples in both oblong and bud stages. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed a high number of functional proteins differentially accumulated in the deyolked versus non-deyolked samples. The most prominent enrichments after the deyolking procedure included processes, functions, and components related to cellular organization, cell cycle, control of replication and translation, and mitochondrial functions. This deyolking procedure improves both qualitative and quantitative proteome analyses and provides an innovative tool in molecular embryogenesis of polylecithal animals, such as fish, amphibians, reptiles, or birds.

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Section: Tube-gel Digestion and Sample Clean Upmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Proteomics Analysis of Early Developmental Stages of Zebrafish Embryos

Purushothaman

Das

Presslauer

et al. 2019

IJMS

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show abstract

“…Proteolytic peptides of this yolk protein could potentially suppress the ionization of other, less abundant proteolytic peptides of nonyolk proteins. 5,6 When using conventional data-dependent acquisitions, the redundant selection of precursor ions from high-abundance peptides to sequence by tandem mass spectrometry (MS/MS) also decreases proteome coverage provided through shotgun proteomics. 7 To overcome this obstacle to in-depth proteomic analyses of zebrafish embryos, a deyolking procedure 1 is done on the embryos to remove as much of the vitellogenin as possible to reduce sample complexity before mass spectrometric analysis thereby increasing the chances of detecting low-abundance proteins.…”

Section: Introductionmentioning

confidence: 99%

A Survey of the Impact of Deyolking on Biological Processes Covered by Shotgun Proteomic Analyses of Zebrafish Embryos

et al. 2015

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Deyolking, the removal of the most abundant protein from the zebrafish (Danio rerio) embryo, is a common technique for in-depth exploration of proteome-level changes in vivo due to various environmental stressors or pharmacological impacts during embryonic stage of development. However, the effect of this procedure on the remaining proteome has not been fully studied. Here, we report a label-free shotgun proteomics survey on proteome coverage and biological processes that are enriched and depleted as a result of deyolking. Enriched proteins are involved in cellular energetics and development pathways, specifically implicating enrichment related to mitochondrial function. Although few proteins were removed completely by deyolking, depleted molecular pathways were associated with calcium signaling and signaling events implicating immune system response.

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“…The final dehydration step was performed using 100 μL acetonitrile. Trypsinization (1.5 μg trypsin) was performed at 37 °C for 16 h. The digested peptides were centrifuged at 6000 × g for 10 min to collect the supernatant and stored at -20 °C (protocol modified from [17]. The samples were lyophilized and 30 μL of the dissolution buffer (0.5 M TEAB, pH 8.5) was added to each sample.…”

Section: Tube-gel Digestion and Sample Clean Upmentioning

confidence: 99%

“…The overwhelming occurrence of vitellogenins -yolk proteins -is a limiting factor in a polylecithal embryo, such as in zebrafish, as it hinders global identification of less abundant proteins using mass spectrometry-based techniques [4,16]. Proteolytic peptides of yolk proteins can potentially subdue the ionization of the less abundant proteolytic peptides of non-yolk proteins [17,18]. Consequently, abundant yolk proteins can potentially interfere with the identification of cellular proteins, although the degree of such the interference is unknown.…”

Section: Introductionmentioning

confidence: 99%

An improved deyolking procedure facilitates the proteomics analysis of early developmental stages of zebrafish embryos

Purushothaman

Presslauer

Das

et al. 2019

Preprint

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Background: Zebrafish is a well-recognised organism for investigating vertebrate development and human diseases. However, the data on zebrafish proteome are scarce, particularly during embryogenesis. This is mostly due to the overwhelming abundance of egg yolk proteins, which tend to mask the detectable presence of less abundant proteins. Results: In this study, we developed an efficient procedure to reduce the amount of yolk in zebrafish early embryos to improve the LC-MS-based shotgun proteomics analysis. We demonstrated that the deyolking procedure resulted in a greater number of proteins being identified. This protocol resulted in approximately two-fold increase in the number of proteins identified in deyolked samples at cleavage stages, and the number of identified proteins increased greatly by 3-4 times compared to non-deyolked samples in both oblong and bud stages. Gene Ontology and KEGG analysis revealed a high number of functional proteins differentially accumulated in the deyolked versus non-deyolked samples. The most prominent enrichments after the deyolking procedure included processes, functions and components related to cellular organization, cell cycle, control of replication and translation, and mitochondrial functions. Conclusion: This deyolking procedure improves both qualitative and quantitative proteome analyses and provides an innovative tool in molecular embryogenesis of polylecithal animals, such as fish, amphibians, reptiles, or birds.

show abstract

Response of peptide intensity to concentration in ESI-MS-based proteome

Cited by 3 publications

References 36 publications

Proteomics Analysis of Early Developmental Stages of Zebrafish Embryos

Proteomics Analysis of Early Developmental Stages of Zebrafish Embryos

A Survey of the Impact of Deyolking on Biological Processes Covered by Shotgun Proteomic Analyses of Zebrafish Embryos

An improved deyolking procedure facilitates the proteomics analysis of early developmental stages of zebrafish embryos

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