Most cited article - PubMed ID 23590590
Suppression of peptide sample losses in autosampler vials
PURPOSE: To explore the protein compositional variability of IVF media and identify sources of undeclared contaminants that interfere with the detection of embryo-derived signals. METHODS: Untargeted and targeted mass spectrometry techniques were used to analyze protein composition in 85 samples of used and unused monophasic IVF media across 13 production lots from two manufacturers. Samples included spent culture media (SCM) from individual embryo cultures, matched controls, and unused (blank) media. Protein-free base media was supplemented with either serum-derived or recombinant human serum albumin (HSA) to evaluate their impact on protein contamination. RESULTS: Proteomic analysis revealed that not only SCM but also unconditioned media contained over 700 undeclared human proteins, many of which are known to be implicated in key cellular pathways. No significant differences were observed between the protein profiles of embryos that reached the blastocyst stage (n = 29) and those arrested at cleavage (n = 24). Instead, protein level variation strongly correlated with media production lot, as shown by targeted analysis of 14 candidate proteins and principal component clustering of 53 SCM samples. Analysis of blank media confirmed substantial lot-to-lot heterogeneity. Supplementation experiments demonstrated that serum-derived HSA introduces undeclared, batch-variable proteins into IVF media, contributing to a non-standardized culture environment and confounding the detection of embryo-derived signals. CONCLUSION: Serum-derived HSA was identified as the primary source of protein contamination in IVF media. This overlooked protein background contributes to variability in clinical culture conditions, undermines the reproducibility of secretome analyses, and complicates the discovery of reliable biomarkers in SCM.
- Keywords
- Embryo culture, Human serum albumin, IVF media composition, Protein biomarkers, Proteomics, Spent culture medium,
- Publication type
- Journal Article MeSH
Poly (ADP-ribose) polymerases (PARPs) are enzymes catalyzing the post-translational addition of chains of ADP-ribose moieties to proteins. In most eukaryotic cells, their primary protein targets are involved in DNA recombination, repair, and chromosome maintenance. Even though this group of enzymes is quite common in both eukaryotes and prokaryotes, no PARP homologs have been described so far in ascomycetous yeasts, leaving their potential roles in this group of organisms unexplored. Here, we characterize Pyl1 protein of Yarrowia lipolytica as the first candidate of PARP in yeasts. We show that the expression of PYL1 gene is increased in mutants lacking either subunit of telomerase and identified several of its candidate protein targets in vivo. We demonstrate that Pyl1p is a functional PARP that undergoes auto-PARylation and PARylates YlKu70/80 complex. We also show that overexpression of PYL1 in Y. lipolytica cells results in dissociation of YlKu80 from telomeres in vivo, supporting the role of Pyl1p in telomere protection and maintenance. Based on our observations, we propose Pyl1p and its homologs identified in other yeast species represent a distinct class of PARPs, thus substantiating a more detailed investigation of their roles in these organisms.
- MeSH
- Fungal Proteins * metabolism genetics chemistry MeSH
- Telomere Homeostasis * MeSH
- Poly(ADP-ribose) Polymerases * metabolism genetics chemistry MeSH
- Telomerase metabolism genetics MeSH
- Telomere * metabolism MeSH
- Yarrowia * enzymology genetics MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Fungal Proteins * MeSH
- Poly(ADP-ribose) Polymerases * MeSH
- Telomerase MeSH
Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease often associated with underlying inflammatory bowel disease (IBD). This study investigates how PSC predisposes individuals to altered inflammatory immune responses compared with IBD alone. A case-control study was conducted with a cohort of 75 patients, including 16 with PSC (14 with concomitant IBD), 39 with IBD alone, and 20 controls. Serum bile acid profile, proteomic analysis, and immune-related gene expression in the colon tissue were examined. Colonic tissue from PSC patients exhibited up-regulation of immune regulation and inflammatory signaling mRNA markers, including LGR5, IL-8, CCL2, COX2, TWIST1, and SNAIL. Additionally, PSC patients displayed a distinct proinflammatory serum proteomic signature and moderate elevation of some bile acids, such as glycochenodeoxycholic acid (GCDCA). Co-incubation of human-derived monocytes with GCDCA partially replicated the inflammatory profile observed in PSC. These findings suggest that circulating bile acids modulate the peripheral immune system proinflammatory response, contributing to the unique PSC phenotype.
- Keywords
- GCDCA, IBD, PSC, bile acids, immune response,
- MeSH
- Adult MeSH
- Inflammatory Bowel Diseases * immunology complications blood genetics MeSH
- Colon metabolism immunology MeSH
- Middle Aged MeSH
- Humans MeSH
- Monocytes immunology metabolism MeSH
- Proteomics methods MeSH
- Cholangitis, Sclerosing * immunology blood complications genetics MeSH
- Case-Control Studies MeSH
- Bile Acids and Salts * blood immunology MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Bile Acids and Salts * MeSH
Anaplastic Large Cell Lymphoma (ALCL) is an aggressive T-cell lymphoma affecting children and young adults. About 30% of patients develop therapy resistance therefore new precision medicine drugs are highly warranted. Multiple rounds of structure-activity optimization of Caffeic Acid Phenethyl Ester have resulted in CM14. CM14 causes upregulation of genes involved in oxidative stress response and downregulation of DNA replication genes leading to G2/M arrest and subsequent apoptosis induction. In accordance with this, an unbiased proteomics approach, confocal microscopy and molecular modeling showed that TUBGCP2, member of the centrosomal γ-TuRC complex, is a direct interaction partner of CM14. CM14 overcomes ALK inhibitor resistance in ALCL and is also active in T-cell Acute Lymphoblastic Leukemia and Acute Myeloid Leukemia. Interestingly, CM14 also induced cell death in docetaxel-resistant prostate cancer cells thus suggesting an unexpected role in solid cancers. Thus, we synthesized and thoroughly characterized a novel TUBGCP2 targeting drug that is active in ALCL but has also potential for other malignancies.
- MeSH
- Apoptosis drug effects MeSH
- Centrosome * drug effects metabolism MeSH
- Drug Resistance, Neoplasm drug effects MeSH
- Phenylethyl Alcohol * pharmacology analogs & derivatives chemistry MeSH
- Caffeic Acids * pharmacology chemistry MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- Antineoplastic Agents * pharmacology chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- caffeic acid phenethyl ester MeSH Browser
- Phenylethyl Alcohol * MeSH
- Caffeic Acids * MeSH
- Antineoplastic Agents * MeSH
Faithful meiotic segregation requires pairwise alignment of the homologous chromosomes and their synaptonemal complex (SC) mediated stabilization. Here, we investigate factors that promote and coordinate these events during C. elegans meiosis. We identify BRA-2 (BMP Receptor Associated family member 2) as an interactor of HIM-17, previously shown to promote double-strand break formation. We found that loss of bra-2 impairs synapsis elongation without affecting homolog recognition, chromosome movement or SC maintenance. Epistasis analyses reveal previously unrecognized activities for HIM-17 in regulating homolog pairing and SC assembly in a partially overlapping manner with BRA-2. We show that removing bra-2 or him-17 restores nuclear clustering, recruitment of PLK-2 at the nuclear periphery, and abrogation of ectopic synapsis in htp-1 mutants, suggesting intact CHK-2-mediated signaling and presence of a barrier that prevents SC polymerization in the absence of homology. Our findings shed light on the regulatory mechanisms ensuring faithful pairing and synapsis.
- MeSH
- Caenorhabditis elegans * genetics metabolism cytology MeSH
- Meiosis * genetics physiology MeSH
- Mutation MeSH
- Chromosome Pairing * genetics MeSH
- Cell Cycle Proteins * metabolism genetics MeSH
- Caenorhabditis elegans Proteins * metabolism genetics MeSH
- Synaptonemal Complex metabolism genetics MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Htp-1 protein, C elegans MeSH Browser
- Cell Cycle Proteins * MeSH
- Caenorhabditis elegans Proteins * MeSH
Microbore columns with a 1.0 mm inner diameter (i.d.) have gained popularity in microflow liquid chromatography-mass spectrometry (LC-MS) workflows for exploratory proteomics applications due to their high throughput, robustness, and reproducibility. However, obtaining highly efficient separation using these columns remains unachievable, primarily due to significant radial flow heterogeneity caused by uneven particle packing density across the column cross-section. In this study, we evaluated the integration of a 1.5 mm i.d. column, which offers greater packing uniformity and reduced radial flow dispersion, into a microflow LC-MS setup for bottom-up proteomics analysis. The performance of the 1.5 mm i.d. column was compared with that of the 1.0 mm i.d. column using protein samples of varying complexity. The results demonstrate that 1.5 mm i.d. columns provide superior chromatographic separation and better compatibility with conventional-flow LC systems, yielding higher reproducibility and comparable protein and peptide identifications to the 1.0 mm i.d. columns at higher sample amounts. These findings suggest that 1.5 mm i.d. columns could be a suitable alternative to 1.0 mm i.d. columns for microflow LC-MS/MS proteomic analysis, particularly in laboratories with only conventional-flow LC systems.
- Publication type
- Journal Article MeSH
Cyclic electron transport around photosystem I (PSI) is essential for the protection of the photosynthetic apparatus in plants under diverse light conditions. This process is primarily mediated by Proton Gradient Regulation 5 protein/Proton Gradient Regulation 5-like photosynthetic phenotype 1 protein (PGR5/PGRL1) and NADH dehydrogenase-like complex (NDH). In angiosperms, NDH interacts with two PSI complexes through distinct monomeric antennae, LHCA5 and LHCA6, which is crucial for its higher stability under variable light conditions. This interaction represents an advanced evolutionary stage and offers limited insight into the origin of the PSI-NDH supercomplex in evolutionarily older organisms. In contrast, the moss Physcomitrium patens (Pp), which retains the lhca5 gene but lacks the lhca6, offers a glimpse into an earlier evolutionary stage of the PSI-NDH supercomplex. Here we present structural evidence of the Pp PSI-NDH supercomplex formation by single particle electron microscopy, demonstrating the unique ability of Pp to bind a single PSI in two different configurations. One configuration closely resembles the angiosperm model, whereas the other exhibits a novel PSI orientation, rotated clockwise. This structural flexibility in Pp is presumably enabled by the variable incorporation of LHCA5 within PSI and is indicative of an early evolutionary adaptation that allowed for greater diversity at the PSI-NDH interface. Our findings suggest that this variability was reduced as the structural complexity of the NDH complex increased in vascular plants, primarily angiosperms. This study not only clarifies the evolutionary development of PSI-NDH supercomplexes but also highlights the dynamic nature of the adaptive mechanisms of plant photosynthesis.
- Keywords
- LHCA5, PSI‐NDH supercomplex, Physcomitrium patens, cyclic electron transport, single particle analysis, transmission electron microscopy,
- MeSH
- Photosynthesis MeSH
- Photosystem I Protein Complex * metabolism genetics MeSH
- Bryopsida * genetics metabolism MeSH
- NADH Dehydrogenase metabolism genetics MeSH
- Plant Proteins * metabolism genetics MeSH
- Light-Harvesting Protein Complexes metabolism genetics chemistry MeSH
- Electron Transport MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Photosystem I Protein Complex * MeSH
- NADH Dehydrogenase MeSH
- Plant Proteins * MeSH
- Light-Harvesting Protein Complexes MeSH
Actomyosin contractility represents an ancient feature of eukaryotic cells participating in many developmental and homeostasis events, including tissue morphogenesis, muscle contraction and cell migration, with dysregulation implicated in various pathological conditions, such as cancer. At the molecular level, actomyosin comprises actin bundles and myosin motor proteins that are sensitive to posttranslational modifications like phosphorylation. While the molecular components of actomyosin are well understood, the coordination of contractility by extracellular and intracellular signals, particularly from cellular signalling pathways, remains incompletely elucidated. This study focuses on WNT/planar cell polarity (PCP) signalling, previously associated with actomyosin contractility during vertebrate neurulation. Our investigation reveals that the main cytoplasmic PCP proteins, Prickle and Dishevelled, interact with key actomyosin components such as myosin light chain 9 (MLC9), leading to its phosphorylation and localized activation. Using proteomics and microscopy approaches, we demonstrate that both PCP proteins actively control actomyosin contractility through Rap1 small GTPases in relevant in vitro and in vivo models. These findings unveil a novel mechanism of how PCP signalling regulates actomyosin contractility through MLC9 and Rap1 that is relevant to vertebrate neurulation.
- Keywords
- MDCK cells, Xenopus embryos, actomyosin contractility, neurulation, planar cell polarity, vertebrates,
- MeSH
- Actomyosin * metabolism MeSH
- Phosphorylation MeSH
- Myosin Light Chains metabolism MeSH
- Humans MeSH
- Mice MeSH
- Neurulation * MeSH
- Vertebrates metabolism MeSH
- Cell Polarity * MeSH
- Dishevelled Proteins metabolism genetics MeSH
- Signal Transduction MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Actomyosin * MeSH
- Myosin Light Chains MeSH
- Dishevelled Proteins MeSH
Polyglutamylation is a reversible posttranslational modification that is catalyzed by enzymes of the tubulin tyrosine ligase-like (TTLL) family. Here, we found that TTLL11 generates a previously unknown type of polyglutamylation that is initiated by the addition of a glutamate residue to the free C-terminal carboxyl group of a substrate protein. TTLL11 efficiently polyglutamylates the Wnt signaling protein Dishevelled 3 (DVL3), thereby changing the interactome of DVL3. Polyglutamylation increases the capacity of DVL3 to get phosphorylated, to undergo phase separation, and to act in the noncanonical Wnt pathway. Both carboxy-terminal polyglutamylation and the resulting reduction in phase separation capacity of DVL3 can be reverted by the deglutamylating enzyme CCP6, demonstrating a causal relationship between TTLL11-mediated polyglutamylation and phase separation. Thus, C-terminal polyglutamylation represents a new type of posttranslational modification, broadening the range of proteins that can be modified by polyglutamylation and providing the first evidence that polyglutamylation can modulate protein phase separation.
- Keywords
- Dishevelled 3, Noncanonical Wnt Signaling, Polyglutamylation, Protein Condensates, TTLL11,
- MeSH
- Phosphorylation MeSH
- HEK293 Cells MeSH
- Polyglutamic Acid metabolism analogs & derivatives MeSH
- Humans MeSH
- Peptide Synthases * metabolism genetics MeSH
- Protein Processing, Post-Translational * MeSH
- Dishevelled Proteins * metabolism genetics MeSH
- Phase Separation MeSH
- Wnt Signaling Pathway MeSH
- Signal Transduction MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- DVL3 protein, human MeSH Browser
- Polyglutamic Acid MeSH
- Peptide Synthases * MeSH
- Dishevelled Proteins * MeSH
- tubulin polyglutamylase MeSH Browser
Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and deeper proteome coverage is needed for its molecular characterization. We present comprehensive library of targeted mass spectrometry assays specific for TNBC and demonstrate its applicability. Proteins were extracted from 105 TNBC tissues and digested. Aliquots were pooled, fractionated using hydrophilic chromatography and analyzed by LC-MS/MS in data-dependent acquisition (DDA) parallel accumulation-serial fragmentation (PASEF) mode on timsTOF Pro LC-MS system. 16 individual lysates were analyzed in data-independent acquisition (DIA)-PASEF mode. Hybrid library was generated in Spectronaut software and covers 244,464 precursors, 168,006 peptides and 11,564 protein groups (FDR = 1%). Application of our library for pilot quantitative analysis of 16 tissues increased identification numbers in Spectronaut 18.5 and DIA-NN 1.8.1 software compared to library-free setting, with Spectronaut achieving the best results represented by 190,310 precursors, 140,566 peptides, and 10,463 protein groups. In conclusion, we introduce assay library that offers the deepest coverage of TNBC proteome to date. The TNBC library is available via PRIDE repository (PXD047793).
