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

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.