Molecular & Cellular Proteomics最新文献

Maternal-embryo interactions play a critical role in early mammalian development, with extracellular vesicles (EVs) playing a key role in intercellular communication. Recent studies have focused on the mechanisms by which maternal-derived factors, such as RNA, proteins, and metabolites influence gap junctions, EVs, and direct cell-to-cell interactions, contributing to embryonic development. In this study, using a proteomics approach, we investigated the impact of EVs secreted from porcine endometrial cells (pEECs) and their protein cargoes on embryonic development. We characterized EVs isolated from pEECs (pEEC-EVs) during the diestrus stage using a nanoparticle tracking analysis and cryo-transmission electron microscopy. Furthermore, the effects of pEEC-EVs with or without hormone treatment on the in vitro attachment of hatched blastocysts were evaluated. The attachment rate of porcine embryos was significantly higher for pEEC-EVs in the hormone treatment group than the control group (23.0 ± 1.7% vs. 36.9 ± 1.9% for control and pEEC-EVs, respectively). Furthermore, hormone treatment altered the expression of proteins involved in cellular organization, protein transport, and immunity. Proteomic analysis revealed distinct biological processes between groups: control EVs supported cytoskeletal organization and adhesion, while hormone-treated EVs were enriched in protein transport, immune regulation, and stress response pathways. Key signaling pathways, including VEGFA-VEGFR2, focal adhesion, and TGF-β, were modulated, influencing implantation and embryogenesis. EVs play a crucial role in maternal-embryo interactions, optimizing implantation conditions and supporting embryo-derived stem cell establishment. These findings enhance our understanding of EV-mediated communication and suggest potential applications for improving reproductive health and assisted reproductive technologies.

Limited proteolysis combined with mass spectrometry (LiP-MS) facilitates probing structural changes on a proteome-wide scale. This method leverages differences in the proteinase K accessibility of native protein structures to concurrently assess structural alterations for thousands of proteins in situ. Distinguishing different contributions to the LiP-MS signal, such as changes in protein abundance or chemical modifications, from structural protein alterations remains challenging. Here, we present the first comprehensive computational pipeline to infer structural alterations for LiP-MS data using a two-step approach. (1) We remove unwanted variations from the LiP signal that are not caused by protein structural effects and (2) infer the effects of variables of interest on the remaining signal. Using LiP-MS data from three species we demonstrate that this approach outperforms previously employed approaches. Our framework provides a uniquely powerful approach for deconvolving LiP-MS signals and separating protein structural changes from changes in protein abundance, post-translational modifications and alternative splicing. Our approach may also be applied to analyze other types of peptide-centric structural proteomics data, such as FPOP or molecular painting data.

As one of the most important post-translational modifications, glycosylation participates in various cellular activities in organisms and is closely associated with many pathogeneses. It has been reported that glycosylation affects liver, spinal cord, and heart tissue regeneration. The zebrafish fin has become a valuable model due to its high regenerative capacity. The molecular mechanism of regeneration has been a hot research topic in the field for a long time. However, studies on the influence of glycosylation during limb regeneration in zebrafish are relatively scarce. We discovered that O-GlcNAc expression, identified by WGA, was elevated during the regeneration of the injured fin in zebrafish using lectin microarray. This phenomenon is due to the upregulation of the expression of OGT enzymes and elevated O-GlcNAcylation levels. To investigate the effects on the fin regeneration when O-GlcNAcylation changes, we used OSMI-1 or Alloxan unilateral microinjection to decrease O-GlcNAcylation and observed that it prevented the fin regeneration. Conversely, the O-GlcNAcylation was impressed by a unilateral microinjection of Thiamet-G or Glucose into the fin, leading to a stimulation of the fin regeneration. To further understand the role of O-GlcNAcylation in fin regeneration, LC-MS/MS was performed to identify O-GlcNAc-glycoproteins. The results demonstrated that the O-GlcNAc glycoproteins, such as THBS4 and HSPG, were involved in the regulation of zebrafish fin regeneration process and were closely associated with certain biological processes, such as stem cell differentiation, ECM-receptor interaction pathway, tissue remodeling, etc. We demonstrated that O-GlcNAc glycoproteins are crucial for zebrafish fin regeneration, during which OGT promotes the process by upregulating the O-GlcNAcylation levels in the zebrafish fin.

Here, we develop PEPSeek, a web-server based software to allow higher performance in the identification of pathogen-derived epitope candidates detected via mass spectrometry in MHC class I immunopeptidomes. We apply it to human and mouse cell lines infected with either SARS-CoV-2, Listeria monocytogenes or Chlamydia trachomatis, thereby identifying a large number of novel antigens and epitopes that we prove to be recognized by CD8⁺ T cells. In infected cells, we identified antigenic peptide features that suggested how processing and presentation of pathogenic antigens differ between pathogens. The quantitative tools of PEPSeek also helped to define how C. trachomatis infection cycle could impact on the antigenic landscape of the host human cell system, likely reflecting metabolic changes occurred in the infected cells.

Human leukocyte antigen class I (HLA-I) molecules present short peptide sequences from endogenous or foreign proteins to cytotoxic T cells. The low abundance of HLA-I peptides poses significant technical challenges for their identification and accurate quantification. While mass spectrometry (MS) is currently a method of choice for direct system-wide identification of cellular immunopeptidome, there is still a need for enhanced sensitivity in detecting and quantifying tumor specific epitopes. As gas phase separation in data-dependent MS data acquisition (DDA) increased HLA-I peptide detection by up to 50%, here, we aimed to evaluate the performance of data-independent acquisition (DIA) in combination with ion mobility (diaPASEF) for high-sensitivity identification of HLA presented peptides. Our streamlined diaPASEF workflow enabled identification of 11,412 unique peptides from 12.5 million A375 cells and 3,426 8-11mers from as low as 500,000 cells with high reproducibility. By taking advantage of HLA binder-specific in-silico predicted spectral libraries, we were able to further increase the number of identified HLA-I peptides. We applied SILAC-DIA to a mixture of labeled HLA-I peptides, calculated heavy-to-light ratios for 7,742 peptides across 5 conditions and demonstrated that diaPASEF achieves high quantitative accuracy up to 4-fold dilution. Finally, we identified and quantified shared neoantigens in a monoallelic C1R cell line model. By spiking in heavy synthetic peptides, we verified the identification of the peptide sequences and calculated relative abundances for 13 neoantigens. Taken together, diaPASEF analysis workflows for HLA-I peptides can increase the peptidome coverage for lower sample amounts. The sensitivity and quantitative precision provided by DIA can enable the detection and quantification of less abundant peptide species such as neoantigens across samples from the same background.

Although the risk of female infertility increases with advancing age, the underlying mechanisms remain unknown. Exosomes in follicular fluid are suggested to regulate folliculogenesis and influence oocyte quality, potentially playing a critical role in age-related infertility. Elucidating their content could enhance the understanding of the molecular mechanisms associated with female aging-induced infertility. In this study, we explored the proteomic profiles of exosomes derived from human follicular fluid to identify protein signatures associated with infertility in both young and aging women. Despite the lack of significant differences in the morphology and particle size of follicular fluid-derived exosomes between the two groups, proteomic analysis revealed a distinct pattern of differentially expressed proteins (DEPs). DEPs associated with B-cell activation, pathogen invasion, and disrupted metabolic processes were significantly more highly expressed in the aging group than in the young group, indicating their involvement in age-related infertility. In vivo experiments demonstrated that the application of exosomes, particularly those derived from young female mice, facilitated the successful maturation of follicles. Key exosomal proteins, including ENO1, HSP90B1, fetuin-B, C7, and APOC4, were found to be associated with follicular maturation. Furthermore, the PI3K/AKT signaling pathway, which is known to be related to folliculogenesis, was activated by the application of exosomes in aging female mice. This study provides novel insights into the aging-associated protein signatures of follicular fluid-derived exosomes and their potential role in infertility. These findings suggest that aging-related protein signatures in exosomes could contribute to the treatment of age-related infertility.

The Human Proteome Organization (HUPO) Proteomics Standards Initiative (PSI) has been successfully developing guidelines, data formats, and controlled vocabularies for both the field of molecular interaction and that of mass spectrometry for more than 20 years. This review explores some of the ways that the proteomics community has benefitted from the development of community standards and takes a look at some of the tools and resources that have been improved or developed as a result of the work of the HUPO-PSI.

Coronavirus infectious disease 19 (COVID-19) can lead to cardiac complications, yet the molecular mechanisms driving these effects remain unclear. Protein glycosylation is crucial for viral replication, immune response, and organ function and has been found to change in the lungs and liver of COVID-19 patients. However, how COVID-19 impacts cardiac protein glycosylation has not been defined. Our study combined single nuclei transcriptomics, mass spectrometry (MS)-based glycomics, and lectin-based tissue imaging to investigate alterations in N-glycosylation in the human heart post-COVID-19. We identified significant expression differences in glycogenes involved in N-glycan biosynthesis and MS analysis revealed a reduction in high mannose and isomers of paucimannose structures post-infection, with changes in paucimannose directly correlating with COVID-19 independent of comorbidities. Our observations suggest that COVID-19 primes cardiac tissues to alter the glycome at all levels, namely metabolism, nucleotide sugar transport, and glycosyltransferase activity. Given the role of N-glycosylation in cardiac function, this study provides a basis for understanding the molecular events leading to cardiac damage post-COVID-19 and informing future therapeutic strategies to treat cardiac complications resulting from coronavirus infections.

Glycosylation of immunoglobulin G (IgG) is recognized as a key modulator of cellular effector functions. At the same time, an increasing body of evidence underlines the importance of other antibody isotypes, especially IgA and IgM, in pathophysiological conditions. Therefore, methods to efficiently study the complex interplay between isotypes, subclasses, and glycosylation of antibodies during acute and chronic states of inflammation are needed. As a solution, we present an integrated and comprehensive method combining simultaneous affinity enrichment of IgG, IgA, and IgM with a single measurement, glycopeptide-centered LC-MS analysis of all isotypes which provides protein-specific (isotype and subclass), and site-specific N- and O-glycosylation quantitation. A two-protease approach provided individual peptides for each glycosylation site, allowing unambiguous compositional assignment and relative quantitation of glycoforms on the MS¹ level as well as structural confirmation and partial isomer assignment on the MS/MS level. We demonstrate that our methodology can be efficiently applied to large clinical studies revealing differences in antibody glycosylation in women during and after pregnancy, as well as between healthy donors and patients with rheumatoid arthritis. In addition, this showcased the advantages of our method in comprehensiveness and resolution of isotypes, subclasses, and glycosylation sites as well as its precision and robustness.