Elizabeth H. Peuchen

Single Cell Proteomics Using Frog (Xenopus laevis) Blastomeres Isolated from Early Stage Embryos, Which Form a Geometric Progression in Protein Content

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.

Surface-Confined Aqueous Reversible Addition–Fragmentation Chain Transfer (SCARAFT) Polymerization Method for Preparation of Coated Capillary Leads to over 10 000 Peptides Identified from 25 ng HeLa Digest by Using Capillary Zone Electrophoresis-Tandem Mass Spectrometry

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 min separation and improved single-shot analysis. An average of 977 protein groups and 5605 unique peptides were identified from 50 ng of an E. coli digest, and 2158 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 h (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.

Capillary electrophoresis coupled to negative mode electrospray ionization-mass spectrometry using an electrokinetically-pumped nanospray interface with primary amines grafted to the interior of a glass emitter

We demonstrate an electrokinetically pumped sheath flow nanospray interface for capillary electrophoresis coupled to negative mode electrospray mass spectrometry. In this interface, application of an electric field generates electro-osmotic flow at the interior of a glass emitter that is pulled to a 10-20µm inner diameter orifice. Electro-osmotic flow pumps liquid around the distal tip of the separation capillary, ensheathing analyte into the electrospray electrolyte. In negative ion mode, negative potential applied to an untreated glass emitter drives sheath flow away from the emitter orifice, decreasing the stability and efficiency of the spray. In this manuscript, we treat a portion of the interior of the electrospray emitter with 3-aminopropyltrimethoxysilane, which grafts primary amines to the interior. The amines take on a positive charge, which reverses electro-osmosis and generates stable sheath flow to the emitter orifice under negative potential. Negative mode operation is demonstrated by analyzing a metabolite extract from stage 1 Xenopus laevis embryos. Production of the treated emitters was quite reproducible. We evaluated the performance of three emitters using a set of amino acids; the relative standard deviation in peak intensity was 7% for the most intense component.

Sensitive and fast characterization of site-specific protein glycosylation with capillary electrophoresis coupled to mass spectrometry

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.

Cloud IaaS for Mass Spectrometry and Proteomics: On-Demand Coupling of Cloud Computing to Experimental Facilities

While many high performance and high throughput scientific computations have been successfully ported to the cloud, coupling data intensive instruments, experiments and sensors to IaaS remains a challenge. We walk through the process of cloud migrating computations on physical IT infrastructure that are integrated with mass spectrometry instruments. A spotlight is put on the challenges of on-demand coupling of cloud resources to a stochastic scheduled physical experiment sensitive to output IO stability. We also discuss scalability of the application with increasing vCPUs and scheduling multiple independent instances of the application to enable burst scientific capability not viable with limited on-site infrastructure. Finally, we overview planned future work to integrate with more complex cloud distributed resources.

Asymmetric distribution of biomolecules of maternal origin in the Xenopus laevis egg and their impact on the developmental plan

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.

Phosphorylation Dynamics Dominate the Regulated Proteome during Early Xenopus Development

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.