The Development of High-Resolution
13 Proteomic Analyses in Xenopus
Elizabeth Van Itallie and Leonid Peshkin
CONTENTS
13.1. Introduction ............................................................................................................................................................. 197
13.2. The Database ........................................................................................................................................................... 198
13.3. Nomenclature and Gene Symbols ........................................................................................................................... 209
13.4. Proteomics and Cell Biology ................................................................................................................................... 200
13.5. X. laevis versus X. tropicalis .................................................................................................................................... 200
13.6. Developmental Atlas of Protein Expression ............................................................................................................ 201
13.7. Post-Translational Modif cations ............................................................................................................................. 201
13.8. Single-Cell Proteomics ............................................................................................................................................ 202
13.9. Discussion and Future Directions ............................................................................................................................ 203
Acknowledgments ................................................................................................................................................................ 204
Reference ............................................................................................................................................................................. 204
13.1. INTRODUCTION
In many instances, proteins are the fnal product of gene
expression and are responsible for the vast majority of biological processes. Xenopus oocytes and embryos are excellent model systems for research questions in the f elds of
biochemistry, cell biology, and developmental biology. In all
of these areas, the specifc functions and biochemical properties of hundreds of proteins have been characterized in detail.
However, our knowledge of complex cellular processes such
as the mitotic spindle, nuclear localization, and gastrulation,
among so many others, are now at the stage where to make
progress, we need to understand endogenous and perturbed
phenotypes and phenomena at the level of thousands of
proteins simultaneously to parse the intricacies of a biological
system. The amino acid sequence and cellular expression
of a specifc protein do not solely determine its function: a
protein’s function is often regulated by differential posttranslational modifcation, by proteolytic cleavage, and/or by
chemical modifcation (e.g. phosphorylation, acetylation, or
glycosylation) and its subcellular and temporal localization
within the cell. Despite the widely acknowledged importance
of these contextual properties of proteins for cell function and
embryogenesis, the methodologies to study thousands of proteins and their modifcations simultaneously currently suffer
from experimental accessibility and depth of coverage compared to transcriptomics approaches. As a result, the biological knowledge resulting from the application of proteomics to
Xenopus biology is limited, and the systemic vision of how
omics-scale measurement of protein can expand biological
knowledge is largely missing. In addition to highlighting the
advances that have been made in this feld, this chapter also
seeks to (1) educate the reader about proteomics, (2) emphasize the strengths and weaknesses of the Xenopus system with
regard to proteomics, and (3) provide ideas for the future.
Throughout this chapter, “proteomics” is taken to mean mass
spectrometry (MS)-based proteomics unless stated otherwise.
In this chapter we will focus on bottom-up tandem mass
spectrometry-based proteomics in Xenopus research. Brief y,
proteins are digested into peptides, and these peptides are
ionized and sprayed into a mass spectrometer. The peptides
have specifc properties based on their mass and ionic charge.
Peptides are measured frst in their intact state (MS1) and
then again after they are fragmented (MS/MS). The amino
acid sequences of the peptide are then determined by comparing the mass spectrometer measurements and a reference
sequence database of theoretical peptides. These peptide
measurements are then summarized into protein measurements. These steps only result in identifcation. How are
differences in protein concentrations/modif cations under
experimental and control conditions quantif ed? Analogous
to “barcodes” in sequencing, digested peptides from different samples can be covalently labeled with various chemical
groups with identical masses that vary in terms of distribution of heavy isotopes around their structure, that is, isobaric
reporters. All of these reporters have the same mass before
the peptides are fragmented, but after fragmentation, the
masses are different. In this way, it can be determined what
fraction of the signal is attributable to different conditions for
a given peptide. The most common isobaric labeling reagents
are iTRAQ and TMT MS-3. More recently, promising TMTC+ technology has been developed (Sonnett et al., 2018a).
DOI: 10.1201/9781003050230-15
197
13 Proteomic Analyses in Xenopus
Elizabeth Van Itallie and Leonid Peshkin
CONTENTS
13.1. Introduction ............................................................................................................................................................. 197
13.2. The Database ........................................................................................................................................................... 198
13.3. Nomenclature and Gene Symbols ........................................................................................................................... 209
13.4. Proteomics and Cell Biology ................................................................................................................................... 200
13.5. X. laevis versus X. tropicalis .................................................................................................................................... 200
13.6. Developmental Atlas of Protein Expression ............................................................................................................ 201
13.7. Post-Translational Modif cations ............................................................................................................................. 201
13.8. Single-Cell Proteomics ............................................................................................................................................ 202
13.9. Discussion and Future Directions ............................................................................................................................ 203
Acknowledgments ................................................................................................................................................................ 204
Reference ............................................................................................................................................................................. 204
13.1. INTRODUCTION
In many instances, proteins are the fnal product of gene
expression and are responsible for the vast majority of biological processes. Xenopus oocytes and embryos are excellent model systems for research questions in the f elds of
biochemistry, cell biology, and developmental biology. In all
of these areas, the specifc functions and biochemical properties of hundreds of proteins have been characterized in detail.
However, our knowledge of complex cellular processes such
as the mitotic spindle, nuclear localization, and gastrulation,
among so many others, are now at the stage where to make
progress, we need to understand endogenous and perturbed
phenotypes and phenomena at the level of thousands of
proteins simultaneously to parse the intricacies of a biological
system. The amino acid sequence and cellular expression
of a specifc protein do not solely determine its function: a
protein’s function is often regulated by differential posttranslational modifcation, by proteolytic cleavage, and/or by
chemical modifcation (e.g. phosphorylation, acetylation, or
glycosylation) and its subcellular and temporal localization
within the cell. Despite the widely acknowledged importance
of these contextual properties of proteins for cell function and
embryogenesis, the methodologies to study thousands of proteins and their modifcations simultaneously currently suffer
from experimental accessibility and depth of coverage compared to transcriptomics approaches. As a result, the biological knowledge resulting from the application of proteomics to
Xenopus biology is limited, and the systemic vision of how
omics-scale measurement of protein can expand biological
knowledge is largely missing. In addition to highlighting the
advances that have been made in this feld, this chapter also
seeks to (1) educate the reader about proteomics, (2) emphasize the strengths and weaknesses of the Xenopus system with
regard to proteomics, and (3) provide ideas for the future.
Throughout this chapter, “proteomics” is taken to mean mass
spectrometry (MS)-based proteomics unless stated otherwise.
In this chapter we will focus on bottom-up tandem mass
spectrometry-based proteomics in Xenopus research. Brief y,
proteins are digested into peptides, and these peptides are
ionized and sprayed into a mass spectrometer. The peptides
have specifc properties based on their mass and ionic charge.
Peptides are measured frst in their intact state (MS1) and
then again after they are fragmented (MS/MS). The amino
acid sequences of the peptide are then determined by comparing the mass spectrometer measurements and a reference
sequence database of theoretical peptides. These peptide
measurements are then summarized into protein measurements. These steps only result in identifcation. How are
differences in protein concentrations/modif cations under
experimental and control conditions quantif ed? Analogous
to “barcodes” in sequencing, digested peptides from different samples can be covalently labeled with various chemical
groups with identical masses that vary in terms of distribution of heavy isotopes around their structure, that is, isobaric
reporters. All of these reporters have the same mass before
the peptides are fragmented, but after fragmentation, the
masses are different. In this way, it can be determined what
fraction of the signal is attributable to different conditions for
a given peptide. The most common isobaric labeling reagents
are iTRAQ and TMT MS-3. More recently, promising TMTC+ technology has been developed (Sonnett et al., 2018a).
DOI: 10.1201/9781003050230-15
197