For many proteins, aggregation is one part of a structural equilibrium that can occur. Balancing productive aggregation versus pathogenic aggregation that leads to toxicity is critical and known to involve adenosine triphosphate (ATP) dependent action of chaperones and disaggregases. Recently a second activity of ATP was identified, that of a hydrotrope which, independent of hydrolysis, was sufficient to solubilize aggregated proteins in vitro. This novel function of ATP was postulated to help regulate proteostasis in vivo. We tested this hypothesis on aggregates found in Xenopus oocyte nucleoli. Our results indicate that ATP has dual roles in the maintenance of protein solubility. We provide evidence of endogenous hydrotropic action of ATP but show that hydrotropic solubilization of nucleolar aggregates is preceded by a destabilizing event. Destabilization is accomplished through an energy dependent process, reliant upon ATP and one or more soluble nuclear factors, or by disruption of a co-aggregate like RNA.

Single cell analysis is required to understand cellular heterogeneity in biological systems. We propose that single cells (blastomeres) isolated from early stage invertebrate, amphibian, or fish embryos are ideal model systems for the development of technologies for single cell analysis. For these embryos, although cell cleavage is not exactly symmetric, the content per blastomere decreases roughly by half with each cell division, creating a geometric progression in cellular content. This progression forms a ladder of single-cell targets for the development of successively higher sensitivity instruments. In this manuscript, we performed bottom-up proteomics on single blastomeres isolated by microdissection from 2-, 4-, 8-, 16-, 32- and 50-cell Xenopus laevis (African clawed frog) embryos. Over 1,400 protein groups were identified in single-run reversed-phase liquid chromatography-electrospray ionization-tandem mass spectrometry from single balstomeres isolated from a 16-cell embryo. When the mass of yolk-free proteins in single blastomeres decreased from ~0.8 μg (16-cell embryo) to ~0.2 μg (50-cell embryo), the number of protein group identifications declined from 1,466 to 644. Around 800 protein groups were quantified across four blastomeres isolated from a 16-cell embryo. By comparing the protein expression among different blastomeres, we observed that the blastomere-to-blastomere heterogeneity in 8-, 16-, 32- and 50-cell embryos increases with development stage, presumably due to cellular differentiation. These results suggest that comprehensive quantitative proteomics on single blastomeres isolated from these early stage embryos can provide valuable insights into cellular differentiation and organ development.

A surface-confined aqueous reversible addition-fragmentation chain transfer (SCARAFT) polymerization method was developed to coat capillaries for use in capillary zone electrophoresis (CZE). SCARAFT polymerization primarily takes place on the inner surface of the capillary instead of in solution, which greatly improves the homogeneity of the coating. Capillaries treated with this coating produced an electroosmotic mobility of 2.8 ± 0.2 × 10−6 cm2·V−1·s−1 (N=3), which is roughly an order of magnitude lower than that of commercial linear polyacrylamide (LPA) coated capillaries. Coated capillaries were evaluated for bottom-up proteomic analysis using CZE. The very low electroosmotic mobility results in a 200-minute separation and improved single-shot analysis. An average of 977 protein groups and 5,605 unique peptides were identified from 50 ng of an E. coli digest and 2,158 protein groups and 10,005 peptides were identified from 25 ng of a HeLa digest using single-shot analysis with a SCARAFT-acrylamide capillary coupled to a Q Exactive HF mass spectrometer. The coating is stable. A single capillary was used for over 200 hours (8.4 days) of continuous operation. RSD in migration time was between 2 and 3% for selected ion electropherograms (SIEs) generated for six ions; median theoretical plate counts ranged from 240,000 to 600,000 for these SIEs. Various types of coatings could be prepared by simply changing the functional vinyl monomers in the polymerization mixture. Positively charged coatings using direct attachment and formation of a block-copolymer were prepared and demonstrated for the separation of mixtures of intact proteins.

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.

The earliest stages of animal development are largely controlled by changes in protein phosphorylation mediated by signaling pathways and cyclin-dependent kinases. In order to decipher these complex networks and to discover new aspects of regulation by this post-translational modification, we undertook an analysis of the X. laevis phosphoproteome at seven developmental stages beginning with stage VI oocytes and ending with two-cell embryos. Concurrent measurement of the proteome and phosphoproteome enabled measurement of phosphosite occupancy as a function of developmental stage. We observed little change in protein expression levels during this period. We detected the expected phosphorylation of MAP kinases, translational regulatory proteins, and subunits of APC/C that validate the accuracy of our measurements. We find that more than half the identified proteins possess multiple sites of phosphorylation that are often clustered, where kinases work together in a hierarchical manner to create stretches of phosphorylated residues, which may be a means to amplify signals or stabilize a particular protein conformation. Conversely, other proteins have opposing sites of phosphorylation that seemingly reflect distinct changes in activity during this developmental timeline.

Capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) is attracting renewed attention for proteomic and metabolomic analysis. An important reason for this interest is the maturation and commercialization of interfaces for coupling CZE with ESI-MS. One of these interfaces is an electrokinetically pumped sheath flow nanospray interface developed by the Dovichi group, in which a very low sheath flow is generated based on electroosmosis within a glass emitter. CMP Scientific has commercialized this interface as the EMASS-II ion source. In this work, we compare the performance of the EMASS-II ion source with our in-house system. The performance of the systems is equivalent. We also couple the EMASS-II ion source with a PrinCE Next|480 capillary electrophoresis autosampler and an Orbitrap mass spectrometer, and analyzed these system’s performance with in terms of sensitivity, reproducibility, and separation performance for separation of tryptic digests, intact proteins, and amino acids. The system produced reproducible analysis of BSA digest; the RSDs of peptide intensity and migration time across 24 runs were less than 20% and 6%, respectively. The system produced linear calibration curve of intensity across a 30-fold range of tryptic digest concentration. The combination of a commercial autosampler and electrospray interface efficiently separated amino acids, peptides, and intact proteins, and only required 5 μL of sample for analysis.

Asymmetric cell division is a ubiquitous feature during the development of higher organisms. Asymmetry is achieved by differential localization or activities of biological molecules such as proteins, and coding and non-coding RNAs. Here, we present subcellular transcriptomic and proteomic analyses along the animal-vegetal axis of Xenopus laevis eggs. More than 98% of the maternal mRNAs could be categorized into four localization profile groups: animal, vegetal, extremely vegetal, and a newly described group of mRNAs that we call extremely animal, which are mRNAs enriched in the animal cortex region. 3′UTRs of localized mRNAs were analyzed for localization motifs. Several putative motifs were discovered for vegetal and extremely vegetal mRNAs, while no distinct conserved motifs for the extremely animal mRNAs were identified, suggesting different localization mechanisms. Asymmetric profiles were also found for proteins, with correlation to those of corresponding mRNAs. Based on unexpected observation of the profiles of the homoeologous genes exd2 we propose a possible mechanism of genetic evolution.

Glycoproteomic analysis requires efficient separation and sensitive detection to enable the comprehensive characterization of glycan heterogeneity. Here, we report the use of capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) with an electrokinetically-pumped nanospray interface for the study of protein glycosylation microheterogeneity. A fast separation was developed that resolved intact glycopeptides generated from standard proteins within ~9min. Differentially terminal-galactosylated and sialylated species with the same glycosylation sites were well resolved. The concentration detection limits for CZE were three times higher than for nanoLC methods; however, a 200-fold smaller injection volume was used in CZE, which reflects the use of an extremely efficient electrospray interface in our CZE-ESI-MS setup. The resulting glycopeptide mass detection limit was two orders of magnitude superior to a nanoLC method. We also observed a 1.5% and 7% average relative standard deviation in peak migration time and glycopeptide relative abundance, and a four order of magnitude linear dynamic range in signal intensity. With CZE-ESI-MS, 40 haptoglobin glycopeptides were identified from roughly 40 fmol of digest.