Optimization and comparison of bottom-up proteomic sample preparation for early-stage Xenopus laevis embryos

Abstract

Xenopus laevis is an important model organism in developmental biology. While there is a large literature on changes in the organism’s transcriptome during development, the study of its proteome is at an embryonic state. Several papers have been published recently that characterize the proteome of Xenopus laevis eggs and early stage embryos; however, proteomic sample preparation optimizations have not been reported. Sample preparation is challenging because a large fraction (~90% by weight) of the egg or early stage embryo is yolk. We compared three common protein extraction buffer systems, mammalian Cell-PE LB™ lysing buffer (NP40), sodium dodecyl sulfate (SDS), and 8M urea, in terms of protein extraction efficiency and protein identifications. SDS extracts contained the highest concentration of proteins, but this extract was dominated by a high concentration of yolk proteins. In contrast, NP40 extracts contained ~30% of the protein concentration as SDS extracts, but excelled in discriminating against yolk proteins, which resulted in more protein and peptide identifications. We then compared digestion methods using both SDS and NP40 extraction methods with one-dimensional reverse phase liquid chromatography tandem mass spectrometry (RPLC-MS/MS). NP40 coupled to a filter-aided sample preparation (FASP) procedure produced nearly twice the number of protein and peptide identifications compared to alternatives. When NP40-FASP samples were subjected to two-dimensional RPLC-ESI-MS/MS, a total of 5171 proteins and 38,885 peptides were identified from single stage of embryos (stage 2), increasing the number of protein identifications by 23% in comparison to other traditional protein extraction methods.

Proteomic Characterization of the Neural Ectoderm Fated Cell Clones in the Xenopus laevis Embryo by High-Resolution Mass Spectrometry

“…It is worth noting that identification of ~2400 proteins from 1 μ g peptides analyzed per run per fraction (5 μ g total measured across 5 fractions) using sucrose-gradient-based yolk depletion in this study indicates sensitivity improvement over a recently optimized protocol, in which the use of mammalian Cell-PE LB lysing buffer (NP40) and filter-aided protein sample processing (FASP) enabled the identification of 4957 proteins by analyzing 30 μ g of peptide mixture across 15 fractions with each measured in technical triplicate. 41 …”

Exosomal trafficking in Xenopus development

“…Both the proteomics of Xenopus and the cell biology of exosomes are in their infancy, and both fields await development and standardization of experimental approaches. Although exosome‐rich fluid compartments in the Xenopus embryo can be efficiently harvested without contaminating yolk or cytosolic proteins, preparation of Xenopus samples for mass spectroscopic proteomic analyses is usually tailored to deal with the overwhelming presence of yolk (Peuchen et al, ). Thus, for comparison between studies, it may become necessary to adopt protocols optimized for yolk‐free preparations enriched in membrane proteins.…”

Capillary electrophoresis coupled to negative mode electrospray ionization-mass spectrometry using an electrokinetically-pumped nanospray interface with primary amines grafted to the interior of a glass emitter

“…Xenopus embryos were fertilized, collected, and processed using published protocols [20, 21]. Embryos are collected at development stage 1.…”