Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Baxi, Aparna B.; Pade, Leena R.; Nemes, Péter

doi:10.3791/63586

Cited by 3 publications

(2 citation statements)

References 57 publications

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“…We studied the model with multiplexing barcoding quantification. For this portion of the study, the D11 lineage was selected, because its descendent cells form tight clusters in the embryo that can be accurately dissected following our established protocols Figure presents the fluorescent labeling of the clone in the neurula (stage 13) embryo.…”

Section: Resultsmentioning

confidence: 99%

Dilute to Enrich for Deeper Proteomics: A Yolk-Depleted Carrier for Limited Populations of Embryonic (Frog) Cells

Pade,

Lombard-Banek,

et al. 2023

J. Proteome Res.

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Abundant proteins challenge deep mass spectrometry (MS) analysis of the proteome. Yolk, the source of food in many developing vertebrate embryos, complicates chemical separation and interferes with detection. We report here a strategy that enhances bottom-up proteomics in yolk-laden specimens by diluting the interferences using a yolk-depleted carrier (YODEC) proteome via isobaric multiplexing quantification. This method was tested on embryos of the South African Clawed Frog (Xenopus laevis), where a >90% yolk proteome content challenges deep proteomics. As a proof of concept, we isolated neural and epidermal fated cell clones from the embryo by dissection or fluorescence-activated cell sorting. Compared with the standard multiplexing carrier approach, YODEC more than doubled the detectable X. laevis proteome, identifying 5,218 proteins from D11 cell clones dissected from the embryo. Ca. ∼80% of the proteins were quantified without dropouts in any of the analytical channels. YODEC with high-pH fractionation quantified 3,133 proteins from ∼8,000 V11 cells that were sorted from ca. 2 embryos (1.5 μg total, or 150 ng yolk-free proteome), marking a 15-fold improvement in proteome coverage vs the standard proteomics approach. About 60% of these proteins were only quantifiable by YODEC, including molecular adaptors, transporters, translation, and transcription factors. While this study was tailored to limited populations of Xenopus cells, we anticipate the approach of "dilute to enrich" using a depleted carrier proteome to be adaptable to other biological models in which abundant proteins challenge deep MS proteomics.

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Section: Resultsmentioning

confidence: 99%

Dilute to Enrich for Deeper Proteomics: A Yolk-Depleted Carrier for Limited Populations of Embryonic (Frog) Cells

Pade,

Lombard-Banek,

et al. 2023

J. Proteome Res.

Self Cite

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show abstract

“…We studied the model with multiplexing barcoding quantification. For this portion of the study, we selected the D11 lineage, because these descendent cells form tight clusters in the embryo that can be accurately dissected following our established protocols 40 . Figure 1 presents the fluorescent labeling of the clone in the neurula (stage 13) embryo.…”

Section: Yodec Is Convenient and Scalablementioning

confidence: 99%

Dilute to Enrich for Deeper Proteomics: A Yolk-Depleted Carrier for Limited Populations of Embryonic (Frog) Cells

Pade,

Lombard-Banek,

et al. 2023

Preprint

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Abundant proteins in biological systems challenge deep mass spectrometry (MS) analysis of the proteome. Yolk, the source of food in many developing vertebrate embryos, complicates chemical separation and interferes with MS signal detection, identification, and quantification. We here report a strategy that enhances bottom-up proteomics in yolk-laden biological systems by diluting out the interferences using a YOlk-DEpleted Carrier proteome (YODEC) via isobaric multiplexing quantification. This mechanism was validated through studies comparing the proteomes of the standard HeLa and South African clawed frog (Xenopus laevis) embryos, where a >90% yolk proteome has historically challenged deep proteomics. The proteome was digested and barcoded before mixing with an isobarically tagged diluent proteome. This diluent carrier was prepared by depleting embryonic tissue of yolk platelets. Compared to the standard multiplexing carrier approach, YODEC more than doubled the detectable X. laevis proteome, allowing for the identification of ~4,200 proteins. Ca. ~80% of the identified proteins were quantified without dropouts in any of the analytical channels. As proof of concept, we applied the strategy to quantify the proteome in cell clones that were isolated from the neurula-stage embryo by dissection or fluorescence-activated cell sorting. YODEC boosted the depth of the measured proteome from ~8,000 cells by 5–15 fold, enabling the detection of molecular adaptors, transporters, translation, and transcription factors. While YODEC was tailored for Xenopus tissues in this study, we anticipate the approach of ‘dilute to enrich’ to also be adaptable to other biological models where abundant proteins challenge the depth of MS proteomics.

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An automated spray-capillary platform for the microsampling and CE-MS analysis of picoliter- and nanoliter-volume samples

Huang

Guo

et al. 2023

Anal Bioanal Chem

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Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Cited by 3 publications

References 57 publications

Dilute to Enrich for Deeper Proteomics: A Yolk-Depleted Carrier for Limited Populations of Embryonic (Frog) Cells

Dilute to Enrich for Deeper Proteomics: A Yolk-Depleted Carrier for Limited Populations of Embryonic (Frog) Cells

Dilute to Enrich for Deeper Proteomics: A Yolk-Depleted Carrier for Limited Populations of Embryonic (Frog) Cells

An automated spray-capillary platform for the microsampling and CE-MS analysis of picoliter- and nanoliter-volume samples

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