202
Xenopus
for the two isoforms, and the pattern is also very similar
for Arg1. Arginase catalyzes the hydrolysis of arginine to
ornithine and urea. This kind of discrepancy was anticipated
by the many early studies postulating translational control,
in which stored mRNA is regulated post-transcriptionally.
Now this phenomenon can be studied on a case-by-case
basis with this genome-wide dataset. What is perhaps most
striking about mRNA-protein discordance is that it is relatively rare even in this comprehensive data set.
13.7. POST-TRANSLATIONAL MODIFICATIONS
Since many processes in biology are controlled by post-translational protein modifcation, quantitative measurement of
post-translational modifcations is perhaps the most important contribution that mass spectrometry-based proteomics
can make. Widely used RNA-sequencing methods do not
inform at all on the post-translational status of proteins. The
strengths of Xenopus as a system are particularly well suited
to studies of post-translational modif cations. As mentioned
earlier, deep quantitative measurement of any post-translational modifcation requires an experimental enrichment for
peptides that are post-translationally modifed. This means
that the requirement for starting material is at least ten times
higher for studies of post-translational modifcations than for
studies of the underlying protein levels. The ability to collect hundreds or even thousands of embryos from the same
clutch, to experimentally manipulate hundreds of embryos
by injection, and the size of Xenopus eggs and embryos
mean this requirement for material is achievable.
The frst study of post-translational modif cations with
mass spectrometry in Xenopus was a non-quantitative study
cataloging phosphorylation sites on proteins in four early
developmental stages up to the stage 10.5 gastrula (McGivern
et al., 2009), where both conserved and novel phosphorylation sites were catalogued on hundreds of proteins. The f rst
quantitative time series measurement of phosphorylation and
acetylation reported on dynamics of only a few dozen changes
(Peshkin et al., 2015) across early embryogenesis, gastrulation, and neurulation. The number of profled peptides was
quite small because they were measured without enrichment
and ref ected modifcations of such abundant proteins as glycolytic enzymes and histones. Two groups measured phosphorylation with replicates and phosphopeptide enrichment
during very early development: one measured ~3500 phosphorylated sites across the 20 minutes following fertilization
and estimated absolute occupancy—the fraction of a protein
with a specif c modifed residue—for ~500 phosphorylations
(Presler et al., 2017); another measured ~9000 relative phosphorylated sites across the stage VI oocyte through the f rst
cleavage (Peuchen et al., 2016; Peuchen et al., 2017). Both
groups obtained evidence for decreasing proline directed
phosphorylation following fertilization. Presler et al. additionally reported ubiquitinated peptides, and Qu et al. measured the de-N-glycoproteome of the egg and the stage 41
tadpole (Qu et al., 2020).
Presler et al. also reported a novel method for estimating the occupancy of post-translational modif cations with
confdence intervals (2017). The method combined previous approaches that only (1) took advantage of the inherent reciprocal relationship between the dynamics of the
non-phosphorylated version of the phospho-peptide and
the phosphorylated version in cases where phosphorylation
levels change between conditions/timepoints (Olsen et al.,
2010) or (2) used phosphatase treatment to artif cially induce
changes in the phosphorylated version of a phospho-peptide
(Lim et al., 2017) into one method that was applicable for
multiplexed experiments. It also took advantage of a system
of equations that describes the relationships between phosphorylated and non-phosphorylated dynamics to report confdence intervals on the estimation of occupancy. Knowing
the absolute amount of change in a modifcation for a protein either across development or downstream of a perturbation is much more useful for interpretation than having
relative changes only. This is because a f ve-fold relative
change could actually be an increase from 0.2% to 1% occupancy, whereas a two-fold relative change could actually be
an increase from 45% to 90%. Importantly, since estimating occupancy for a signifcant number of phosphorylation
events with confdence requires artifcially inducing changes
with phosphatase treatment, occupancy estimation needs to
be considered in the initial experimental design.
There is extensive unrealized potential for the intersection of the Xenopus system and post-translational modif cations measured by mass spectrometry. The published studies
have utilized the strengths of natural cell cycle arrests of
the stage 6 oocyte and egg for measuring the endogenous
dynamics, but no study to date has included cell cycle
perturbations. The stage 6 oocyte with its low amount of
mitotic phosphorylation could be used to study low-occupancy phosphorylation events that are often unmeasured
at the expense of high-occupancy cell cycle phosphorylations. Phosphorylation, acetylation, methylation, and ubiquitination have never been quantitatively measured after the
frst division. Studies in cell culture models have measured
extensive phosphorylation of proteins involved in regulation
of gene expression (Rigbolt et al., 2011). The importance
of regulated gene expression in development is well understood, but the importance of phosphorylation for the function and regulation of protein expression is less well studied.
Many proteins that do not change in level during development could be regulated by phosphorylation, which makes
this an important next area of investigation in Xenopus.
13.8. SINGLE-CELL PROTEOMICS
Several years ago, our group developed a high-throughput
droplet-microfuidic approach for barcoding the RNA from
individual cells for subsequent analysis by next-generation
sequencing: an inDrop platform that encapsulates cells
into droplets with a lysis buffer, reverse transcription (RT)
reagents, and barcoded oligonucleotide primers (Klein et al.,
Xenopus
for the two isoforms, and the pattern is also very similar
for Arg1. Arginase catalyzes the hydrolysis of arginine to
ornithine and urea. This kind of discrepancy was anticipated
by the many early studies postulating translational control,
in which stored mRNA is regulated post-transcriptionally.
Now this phenomenon can be studied on a case-by-case
basis with this genome-wide dataset. What is perhaps most
striking about mRNA-protein discordance is that it is relatively rare even in this comprehensive data set.
13.7. POST-TRANSLATIONAL MODIFICATIONS
Since many processes in biology are controlled by post-translational protein modifcation, quantitative measurement of
post-translational modifcations is perhaps the most important contribution that mass spectrometry-based proteomics
can make. Widely used RNA-sequencing methods do not
inform at all on the post-translational status of proteins. The
strengths of Xenopus as a system are particularly well suited
to studies of post-translational modif cations. As mentioned
earlier, deep quantitative measurement of any post-translational modifcation requires an experimental enrichment for
peptides that are post-translationally modifed. This means
that the requirement for starting material is at least ten times
higher for studies of post-translational modifcations than for
studies of the underlying protein levels. The ability to collect hundreds or even thousands of embryos from the same
clutch, to experimentally manipulate hundreds of embryos
by injection, and the size of Xenopus eggs and embryos
mean this requirement for material is achievable.
The frst study of post-translational modif cations with
mass spectrometry in Xenopus was a non-quantitative study
cataloging phosphorylation sites on proteins in four early
developmental stages up to the stage 10.5 gastrula (McGivern
et al., 2009), where both conserved and novel phosphorylation sites were catalogued on hundreds of proteins. The f rst
quantitative time series measurement of phosphorylation and
acetylation reported on dynamics of only a few dozen changes
(Peshkin et al., 2015) across early embryogenesis, gastrulation, and neurulation. The number of profled peptides was
quite small because they were measured without enrichment
and ref ected modifcations of such abundant proteins as glycolytic enzymes and histones. Two groups measured phosphorylation with replicates and phosphopeptide enrichment
during very early development: one measured ~3500 phosphorylated sites across the 20 minutes following fertilization
and estimated absolute occupancy—the fraction of a protein
with a specif c modifed residue—for ~500 phosphorylations
(Presler et al., 2017); another measured ~9000 relative phosphorylated sites across the stage VI oocyte through the f rst
cleavage (Peuchen et al., 2016; Peuchen et al., 2017). Both
groups obtained evidence for decreasing proline directed
phosphorylation following fertilization. Presler et al. additionally reported ubiquitinated peptides, and Qu et al. measured the de-N-glycoproteome of the egg and the stage 41
tadpole (Qu et al., 2020).
Presler et al. also reported a novel method for estimating the occupancy of post-translational modif cations with
confdence intervals (2017). The method combined previous approaches that only (1) took advantage of the inherent reciprocal relationship between the dynamics of the
non-phosphorylated version of the phospho-peptide and
the phosphorylated version in cases where phosphorylation
levels change between conditions/timepoints (Olsen et al.,
2010) or (2) used phosphatase treatment to artif cially induce
changes in the phosphorylated version of a phospho-peptide
(Lim et al., 2017) into one method that was applicable for
multiplexed experiments. It also took advantage of a system
of equations that describes the relationships between phosphorylated and non-phosphorylated dynamics to report confdence intervals on the estimation of occupancy. Knowing
the absolute amount of change in a modifcation for a protein either across development or downstream of a perturbation is much more useful for interpretation than having
relative changes only. This is because a f ve-fold relative
change could actually be an increase from 0.2% to 1% occupancy, whereas a two-fold relative change could actually be
an increase from 45% to 90%. Importantly, since estimating occupancy for a signifcant number of phosphorylation
events with confdence requires artifcially inducing changes
with phosphatase treatment, occupancy estimation needs to
be considered in the initial experimental design.
There is extensive unrealized potential for the intersection of the Xenopus system and post-translational modif cations measured by mass spectrometry. The published studies
have utilized the strengths of natural cell cycle arrests of
the stage 6 oocyte and egg for measuring the endogenous
dynamics, but no study to date has included cell cycle
perturbations. The stage 6 oocyte with its low amount of
mitotic phosphorylation could be used to study low-occupancy phosphorylation events that are often unmeasured
at the expense of high-occupancy cell cycle phosphorylations. Phosphorylation, acetylation, methylation, and ubiquitination have never been quantitatively measured after the
frst division. Studies in cell culture models have measured
extensive phosphorylation of proteins involved in regulation
of gene expression (Rigbolt et al., 2011). The importance
of regulated gene expression in development is well understood, but the importance of phosphorylation for the function and regulation of protein expression is less well studied.
Many proteins that do not change in level during development could be regulated by phosphorylation, which makes
this an important next area of investigation in Xenopus.
13.8. SINGLE-CELL PROTEOMICS
Several years ago, our group developed a high-throughput
droplet-microfuidic approach for barcoding the RNA from
individual cells for subsequent analysis by next-generation
sequencing: an inDrop platform that encapsulates cells
into droplets with a lysis buffer, reverse transcription (RT)
reagents, and barcoded oligonucleotide primers (Klein et al.,