Proteins in Tumor-Derived Plasma Extracellular Vesicles Indicate Tumor Origin

Cancer-derived extracellular vesicles (EVs) promote tumorigenesis, premetastatic niche formation, and metastasis via their protein cargo. However, the proteins packaged by patient tumors into EVs cannot be determined in vivo because of the presence of EVs derived from other tissues. We therefore developed a cross-species proteomic method to quantify the human tumor-derived proteome of plasma EVs produced by patient-derived xenografts of four cancer types. Proteomic profiling revealed individualized packaging of novel protein cargo, and machine learning accurately classified the type of the underlying tumor.     

A comparative Proteomics Analysis Identified Differentially Expressed Proteins in Pancreatic Cancer–Associated Stellate Cell Small Extracellular Vesicles

Human pancreatic stellate cells (HPSCs) are an essential stromal component and mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles involved in cell-to-cell communications and are released from stromal cells within PDAC. A detailed comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs) remains a gap in our current knowledge regarding stellate cells and PDAC. We hypothesized there would be differences in sEVs secretion and protein expression that might contribute to PDAC biology. To test this hypothesis, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. We report here our initial observations. First, HPSC cells derived from PDAC tumors secrete a higher volume of sEVs when compared to normal pancreatic stellate cells (HPaStec). Although our data revealed that both normal and tumor-derived sEVs demonstrated no significant biological effect on cancer cells, we observed efficient uptake of sEVs by both normal and cancer epithelial cells. Additionally, intact membrane-associated proteins on sEVs were essential for efficient uptake. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography–tandem mass spectrometry. Most of the 1481 protein groups identified were shared with the exosome database, ExoCarta. Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted p value ≤0.05) between HPSC and HPaStec sEVs. Of note, HPSC sEVs contained dramatically more CSE1L (chromosome segregation 1–like protein), a described marker of poor prognosis in patients with pancreatic cancer. Based on our results, we have demonstrated unique populations of sEVs originating from stromal cells with PDAC and suggest that these are significant to cancer biology. Further studies should be undertaken to gain a deeper understanding that could drive novel therapy.     

Multiomic Metabolic Enrichment Network Analysis Reveals Metabolite–Protein Physical Interaction Subnetworks Altered in Cancer

Metabolism is recognized as an important driver of cancer progression and other complex diseases, but global metabolite profiling remains a challenge. Protein expression profiling is often a poor proxy since existing pathway enrichment models provide an incomplete mapping between the proteome and metabolism. To overcome these gaps, we introduce multiomic metabolic enrichment network analysis (MOMENTA), an integrative multiomic data analysis framework for more accurately deducing metabolic pathway changes from proteomics data alone in a gene set analysis context by leveraging protein interaction networks to extend annotated metabolic models. We apply MOMENTA to proteomic data from diverse cancer cell lines and human tumors to demonstrate its utility at revealing variation in metabolic pathway activity across cancer types, which we verify using independent metabolomics measurements. The novel metabolic networks we uncover in breast cancer and other tumors are linked to clinical outcomes, underscoring the pathophysiological relevance of the findings.     

Functional Diversity and Evolution of the Drosophila Sperm Proteome

Spermatozoa are central to fertilization and the evolutionary fitness of sexually reproducing organisms. As such, a deeper understanding of sperm proteomes (and associated reproductive tissues) has proven critical to the advancement of the fields of sexual selection and reproductive biology. Due to their extraordinary complexity, proteome depth-of-coverage is dependent on advancements in technology and related bioinformatics, both of which have made significant advancements in the decade since the last Drosophila sperm proteome was published. Here, we provide an updated version of the Drosophila melanogaster sperm proteome (DmSP3) using improved separation and detection methods and an updated genome annotation. Combined with previous versions of the sperm proteome, the DmSP3 contains a total of 3176 proteins, and we provide the first label-free quantitation of the sperm proteome for 2125 proteins. The top 20 most abundant proteins included the structural elements α- and β-tubulins and sperm leucyl-aminopeptidases. Both gene content and protein abundance were significantly reduced on the X chromosome, consistent with prior genomic studies of X chromosome evolution. We identified 9 of the 16 Y-linked proteins, including known testis-specific male fertility factors. We also identified almost one-half of known Drosophila ribosomal proteins in the DmSP3. The role of this subset of ribosomal proteins in sperm is unknown. Surprisingly, our expanded sperm proteome also identified 122 seminal fluid proteins (Sfps), proteins originally identified in the accessory glands. We show that a significant fraction of ‘sperm-associated Sfps’ are recalcitrant to concentrated salt and detergent treatments, suggesting this subclass of Sfps are expressed in testes and may have additional functions in sperm, per se. Overall, our results add to a growing landscape of both sperm and seminal fluid protein biology and in particular provides quantitative evidence at the protein level for prior findings supporting the meiotic sex-chromosome inactivation model for male-specific gene and X chromosome evolution.     

Thorough Performance Evaluation of 213 nm Ultraviolet Photodissociation for Top-down Proteomics

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Highlights

• •Analysis of product ions produced by 213 nm UVPD is used to refine database search.
• •A product ion at the N-terminus of Pro, y-2, is observed in 213 nm UVPD spectra.
• •213 nm UVPD provides more complete proteoform characterization than HCD.
• •HCD and 213 nm UVPD are complementary fragmentation methods for proteoforms <30 kDa.

     

Identification of a caleosin associated with hazelnut (Corylus avellana L.) oil bodies

• Caleosins are involved in several cellular and biological processes that are closely associated with the synthesis, degradation and stability of oil bodies (OB).
• Because of the importance and the multiple roles of these OB‐associated proteins, in silico identification of sequences corresponding to putative caleosins in the hazelnut genome has been performed, and the association with seed OB was verified using a proteomic approach.
• Five full‐length sequences (CavCLO‐H1, CavCLO‐H2, CavCLO‐H3, CavCLO‐L1, CavCLO‐L2), belonging to the two groups of caleosins (H and L), have been identified in the hazelnut genome. The number of identified caleosins is in agreement with that previously observed in other plant species, confirming that caleosins comprise small gene families in plants.
• A proteomic approach allowed us to verify only the presence of CavCLO‐H1 in hazelnut OB, suggesting that several members inside this family could have different roles during plant growth and development. In silico analysis also suggests that CavCLO‐H1 may act as a peroxygenase.