Quantitative Proteomics of Xenopus Embryos I, Sample Preparation

Abstract

Xenopus oocytes and embryos are model systems optimally suited for quantitative proteomics. This is due to the availability of large amount of protein material and the ease of physical manipulation. Furthermore, facile in vitro fertilization provides superbly synchronized embryos for cell cycle and developmental stages. Here, we detail protocols developed over the last few years for sample preparation of multiplexed proteomics with TMT-tags followed by quantitative mass spectrometry analysis using the MultiNotch MS3 approach. In this approach, each condition is barcoded with an isobaric tag at the peptide level. After barcoding, samples are combined and the relative abundance of ~100,000 peptides is quantified on a mass spectrometer. High reproducibility of the sample preparation process prior to peptides being tagged and combined is of upmost importance for obtaining unbiased data. Otherwise, differences in sample handling can inadvertently appear as biological changes. We detail and exemplify the application of our sample workflow on an embryonic time-series of ten developmental stages of Xenopus laevis embryos ranging from the egg to stage 35 (just before hatching). Our accompanying paper (Chapter 14 ) details a bioinformatics pipeline to analyze the quality of the given sample preparation and strategies to convert spectra of X. laevis peptides into biologically interpretable data.

Keywords: Development; Mass spectrometry; Multiplexing; Protein dynamics; Proteomics; Sample preparation; TMT; Xenopus laevis; Yolk.

Fig. 1

Principle of multiplexed proteomics. (A) Proteins from multiple conditions (replicates, time points, etc.) are digested and labeled with isobaric tags (e.g., TMT). The distinct colors represent different tags, which serve as barcodes for the sample origin. After tagging, different conditions are mixed and together ionized onto a mass spectrometer. (B) In the MS1 spectrum the isobaric tags have the identical mass, thus peptide peaks from different conditions are indistinguishable. (C) The isolated peptides are fragmented and produce condition-specific reporter ions. The intensities of these reporter ions can be used for relative quantification of the associated protein. The b- and y-ions, which result from breakage of the peptide’s backbone, are used for identification

Fig. 2

Yolk spin out. (A) ~90% of the protein mass in Xenopus embryos are yolk. Shown is a Coomassie-stained gel of a lysed Xenopus egg (left) and egg proteins after removing yolk via centrifugation (right). (B) Eppendorf tube after centrifugation step as detailed in the paper. Yolk and pigments sediment. Solubilized proteins can be removed from the supernatant

Fig. 3

Elution profile of the peptides during prefractionation. The green lines mark the fractions collected at various times starting at 17 min with 42 s increments until the 96th fraction at 84.5 mins (The first three fractions are marked as 1, 2, and 3). The large peak at the beginning corresponds to small molecules

Fig. 4

Schematic representation of concatenation strategy. Each color in the vertical column represents fractions that are combined into a sample, which is analyzed via LC-MS. After the first 12 collections, collect another 12 by following the same scheme using the uncolored wells in the vertical rows

Fig. 5

Parameter setting for TMT-MS3 method

Fig. 5

Parameter setting for TMT-MS3 method