200
Xenopus
and thus maps into the symbol “nodal.” When the pipeline
for such reciprocal BLAST-based gene symbol assignment
was developed and applied to X. laevis (Savova et al., 2017),
17,000 unique human gene symbols matched. Not surprisingly, in X. tropicalis, which has a smaller genome size but
no allo-alleles (allo-alleles are essentially redundant in terms
of genome complexity as assayed by homology to human),
the set of gene models match a similar number of human
gene symbols as for X. laevis.
Even though the Xenopus genome assemblies are in relatively good shape, much remains to be done in terms of gene
annotation and cataloging of expressed protein forms. A
hybrid approach to generating a complete non-redundant set
of reference protein sequences that combines genome-based
and genome-free (mRNA-based) methods is an unresolved
problem. Methods such as ribosome prof ling (RIBO-seq)
or ribosome nascent chain sequencing (RNC-seq) could be
used to identify the set of mRNAs that are actually associated with ribosomes and thereby being translated (Savova
et al., 2017; Zhao et al., 2019) and to identify current unannotated protein coding regions (Verbruggen et al., 2019).
13.4. PROTEOMICS AND CELL BIOLOGY
Xenopus laevis is an important model for studies of cell
biology and, as reported in other chapters in this book, has
been used extensively to understand the cellular regulation
of mitosis, DNA replication, transcription and translation,
RNA and protein localization, and embryogenesis, among
many other processes. Recent advances in proteomics have
enriched our understanding of several cellular processes,
and a few are summarized here.
One elegant study took advantage of the fact that not only is
the Xenopus stage VI oocyte a very large cell, but the nucleus
(germinal vesicle) is also very large. Wühr et al. removed the
germinal vesicle from the oocyte with forceps and measured
the distribution of the proteome between the nucleus and the
cytoplasm (2015). We found that protein localization can be
mostly explained by empirical measurement of native protein size; the majority of large proteins are found exclusively
in either the nucleus or the cytoplasm, and smaller proteins
are found equally distributed between the two. This measurement of native protein size was only possible because of the
ability to make undiluted cell lysates from crushed oocytes.
Molecular weight determined by polypeptide length did not
predict localization well, which is evidence that many proteins natively exist in complexes. In addition to providing
insights into the properties of proteins in undiluted cytoplasm, this work produced an important resource for nuclear
versus cytoplasmic localization of proteins.
Presler et al. used egg activation as a model of the f rst
20 minutes following release from meiotic metaphase arrest
(2017). We detected very few previously unknown degradation events and no examples of protein synthesis. Instead,
we found that expulsion of proteins from the eggs, assumed
to occur via fusion of cortical granules with the cell membrane, constitutes the largest change to protein level during
this period. We were able to compare these changes in absolute concentration amounts because of our previous work
determining the concentration of proteins in the egg (Wühr
et al., 2014). This is an example of using proteomics to reexamine bulk biochemical measurements of the egg and early
embryo with the genomics-era ability to identify and classify the proteins that constitute these changes.
Aff nity purifcation is another proteomics strategy that
has been used by the Xenopus community. Lee et al. used
dissected animal caps to identify novel binding partners of
inner and outer dynein arm subunits in liquid-like organelles
(Dynein Axonemal Particles or DynAPs) before their assembly into cilia (2020). Identifcation of novel protein localization in DynAPs explained why certain proteins that are not
found in motile cilia themselves can still give rise to ciliopathies when they are mutated. Drew et al. used the same animal cap system, and a methodology termed DIF-FRAC, to
identify RNA-binding proteins in epidermal tissues at the
time point when motile cilia have formed (2020). They found
evidence that there is RNA associated with DynAPs which is
consistent with reports of RNA in other cytoplasmic liquidlike organelles. These two papers exemplify taking advantage of the strengths of the Xenopus simple explant system to
enrich for the cellular complexes or tissue of interest. Animal
cap explants can be differentiated into many different tissue
types, and this is a way to overcome the challenges of tissue
type heterogeneity of the embryo while also using a vertebrate animal system (Chang, 2016).
13.5. X. LAEVIS VERSUS X. TROPICALIS
Currently, MS-based proteomics in Xenopus is almost
entirely done in X. laevis and not X. tropicalis, likely due
to the preponderance of laboratories that use X. laevis for
their other experimental approaches. Although X. tropicalis
animals are smaller (oocytes are only half size in diameter),
they have many advantages for proteomics: their development is faster, so reaching more advanced developmental
points is easier; there are many transgenic strains available;
and CRISPR-based genome editing works well (Nakayama
et al., 2014; Naert et al., 2020; Horb et al., 2021). The simplicity of the genome makes bioinformatics easier and should
result in the measurement of more functionally distinct proteins and post-translational modifcations. This is because
not all present peptides can be measured, and the presence
of allo-alleles increases the fraction of redundant peptides;
thus, spectra matching is more complex in X. laevis for the
same level of sample biological complexity. Additionally,
owing to being a true diploid, the complexity of the protein
and peptide mixture is simpler in X. tropicalis than in X. laevis, which improves the eff ciency in both the sample workfow (labeling, fractionation) and the bioinformatics. Being
diploid also means that the genome assembly and annotation
is at a more advanced state in X. tropicalis, so interpretation
of the results is also more robust. In our experiments (unpublished), we see 40% of collected X. tropicalis spectra successfully matched to a sequence, in contrast with X. laevis,
Xenopus
and thus maps into the symbol “nodal.” When the pipeline
for such reciprocal BLAST-based gene symbol assignment
was developed and applied to X. laevis (Savova et al., 2017),
17,000 unique human gene symbols matched. Not surprisingly, in X. tropicalis, which has a smaller genome size but
no allo-alleles (allo-alleles are essentially redundant in terms
of genome complexity as assayed by homology to human),
the set of gene models match a similar number of human
gene symbols as for X. laevis.
Even though the Xenopus genome assemblies are in relatively good shape, much remains to be done in terms of gene
annotation and cataloging of expressed protein forms. A
hybrid approach to generating a complete non-redundant set
of reference protein sequences that combines genome-based
and genome-free (mRNA-based) methods is an unresolved
problem. Methods such as ribosome prof ling (RIBO-seq)
or ribosome nascent chain sequencing (RNC-seq) could be
used to identify the set of mRNAs that are actually associated with ribosomes and thereby being translated (Savova
et al., 2017; Zhao et al., 2019) and to identify current unannotated protein coding regions (Verbruggen et al., 2019).
13.4. PROTEOMICS AND CELL BIOLOGY
Xenopus laevis is an important model for studies of cell
biology and, as reported in other chapters in this book, has
been used extensively to understand the cellular regulation
of mitosis, DNA replication, transcription and translation,
RNA and protein localization, and embryogenesis, among
many other processes. Recent advances in proteomics have
enriched our understanding of several cellular processes,
and a few are summarized here.
One elegant study took advantage of the fact that not only is
the Xenopus stage VI oocyte a very large cell, but the nucleus
(germinal vesicle) is also very large. Wühr et al. removed the
germinal vesicle from the oocyte with forceps and measured
the distribution of the proteome between the nucleus and the
cytoplasm (2015). We found that protein localization can be
mostly explained by empirical measurement of native protein size; the majority of large proteins are found exclusively
in either the nucleus or the cytoplasm, and smaller proteins
are found equally distributed between the two. This measurement of native protein size was only possible because of the
ability to make undiluted cell lysates from crushed oocytes.
Molecular weight determined by polypeptide length did not
predict localization well, which is evidence that many proteins natively exist in complexes. In addition to providing
insights into the properties of proteins in undiluted cytoplasm, this work produced an important resource for nuclear
versus cytoplasmic localization of proteins.
Presler et al. used egg activation as a model of the f rst
20 minutes following release from meiotic metaphase arrest
(2017). We detected very few previously unknown degradation events and no examples of protein synthesis. Instead,
we found that expulsion of proteins from the eggs, assumed
to occur via fusion of cortical granules with the cell membrane, constitutes the largest change to protein level during
this period. We were able to compare these changes in absolute concentration amounts because of our previous work
determining the concentration of proteins in the egg (Wühr
et al., 2014). This is an example of using proteomics to reexamine bulk biochemical measurements of the egg and early
embryo with the genomics-era ability to identify and classify the proteins that constitute these changes.
Aff nity purifcation is another proteomics strategy that
has been used by the Xenopus community. Lee et al. used
dissected animal caps to identify novel binding partners of
inner and outer dynein arm subunits in liquid-like organelles
(Dynein Axonemal Particles or DynAPs) before their assembly into cilia (2020). Identifcation of novel protein localization in DynAPs explained why certain proteins that are not
found in motile cilia themselves can still give rise to ciliopathies when they are mutated. Drew et al. used the same animal cap system, and a methodology termed DIF-FRAC, to
identify RNA-binding proteins in epidermal tissues at the
time point when motile cilia have formed (2020). They found
evidence that there is RNA associated with DynAPs which is
consistent with reports of RNA in other cytoplasmic liquidlike organelles. These two papers exemplify taking advantage of the strengths of the Xenopus simple explant system to
enrich for the cellular complexes or tissue of interest. Animal
cap explants can be differentiated into many different tissue
types, and this is a way to overcome the challenges of tissue
type heterogeneity of the embryo while also using a vertebrate animal system (Chang, 2016).
13.5. X. LAEVIS VERSUS X. TROPICALIS
Currently, MS-based proteomics in Xenopus is almost
entirely done in X. laevis and not X. tropicalis, likely due
to the preponderance of laboratories that use X. laevis for
their other experimental approaches. Although X. tropicalis
animals are smaller (oocytes are only half size in diameter),
they have many advantages for proteomics: their development is faster, so reaching more advanced developmental
points is easier; there are many transgenic strains available;
and CRISPR-based genome editing works well (Nakayama
et al., 2014; Naert et al., 2020; Horb et al., 2021). The simplicity of the genome makes bioinformatics easier and should
result in the measurement of more functionally distinct proteins and post-translational modifcations. This is because
not all present peptides can be measured, and the presence
of allo-alleles increases the fraction of redundant peptides;
thus, spectra matching is more complex in X. laevis for the
same level of sample biological complexity. Additionally,
owing to being a true diploid, the complexity of the protein
and peptide mixture is simpler in X. tropicalis than in X. laevis, which improves the eff ciency in both the sample workfow (labeling, fractionation) and the bioinformatics. Being
diploid also means that the genome assembly and annotation
is at a more advanced state in X. tropicalis, so interpretation
of the results is also more robust. In our experiments (unpublished), we see 40% of collected X. tropicalis spectra successfully matched to a sequence, in contrast with X. laevis,