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

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.