Journal of Proteome Research最新文献

Immunoprecipitation is among the most widely utilized methods in biomedical research, with applications that include the identification of antibody targets and associated proteins. The path to identifying these targets is not straightforward, however, and often requires the use of chemical cross-linking and/or gel electrophoresis to separate targets from an overabundance of immunoglobulin protein. Such experiments are labor intensive and often yield long lists of candidate antibody targets. Here, we describe an unbiased immunoprecipitation-to-mass spectrometry (IP-to-MS) method that relies on a novel protein tag to separate low abundance immunoprecipitated proteins from overwhelmingly abundant immunoglobulins. We demonstrate that the IP-to-MS serotyping workflow is highly reproducible and can be used for the identification of novel, patient-specific antigen targets in multiple disease states. Furthermore, we show that IP-to-MS may outperform conventional methods of antibody detection, including enzyme-linked immunosorbent assay, while also enabling patient stratification beyond what is possible with traditional approaches.

Inhibition of the mitochondrial deubiquitinating (DUB) enzyme USP30 is neuroprotective and presents therapeutic opportunities for the treatment of idiopathic Parkinson’s disease and mitophagy-related disorders. We integrated structural and quantitative proteomics with biochemical assays to decipher the mode of action of covalent USP30 inhibition by a small-molecule containing a cyanopyrrolidine reactive group, USP30-I-1. The inhibitor demonstrated high potency and selectivity for endogenous USP30 in neuroblastoma cells. Enzyme kinetics and hydrogen–deuterium eXchange mass spectrometry indicated that the inhibitor binds tightly to regions surrounding the USP30 catalytic cysteine and positions itself to form a binding pocket along the thumb and palm domains of the protein, thereby interfering its interaction with ubiquitin substrates. A comparison to a noncovalent USP30 inhibitor containing a benzosulfonamide scaffold revealed a slightly different binding mode closer to the active site Cys77, which may provide the molecular basis for improved selectivity toward USP30 against other members of the DUB enzyme family. Our results highlight advantages in developing covalent inhibitors, such as USP30-I-1, for targeting USP30 as treatment of disorders with impaired mitophagy.

Since late 2021, Omicron variants have dominated the epidemiological scenario as the most successful severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sublineages, driving new and breakthrough infections globally over the past two years. In this study, we investigated for the first time the host salivary response of COVID-19 patients infected with Omicron variants (BA.1, BA.2, and BA.4/5) by using an untargeted four-dimensional data-independent acquisition (4D-DIA)-based proteomics approach. We identified 137 proteins whose abundance levels differed between the COVID-19 positive and negative groups. Salivary signatures were mainly enriched in ribosomal proteins, linked to mRNAviral translation, protein synthesis and processing, immune innate, and antiapoptotic signaling. The higher abundance of 14-3-3 proteins (YWHAG, YWHAQ, YWHAE, and SFN) in saliva, first reported here, may be associated with increased infectivity and improved viral replicative fitness. We also identified seven proteins (ACTN1, H2AC2, GSN, NDKA, CD109, GGH, and PCYOX) that yielded comprehension into Omicron infection and performed outstandingly in screening patients with COVID-19 in a hospital setting. This panel also presented an enhanced anti-COVID-19 and anti-inflammatory signature, providing insights into disease severity, supported by comparisons with other proteome data sets. The salivary signature provided valuable insights into the host’s response to SARS-CoV-2 Omicron infection, shedding light on the pathophysiology of COVID-19, particularly in cases associated with mild disease. It also underscores the potential clinical applications of saliva for disease screening in hospital settings. Data are available via ProteomeXchange with the identifier PXD054133.

The National Cancer Institute’s Clinical Proteomics Tumor Analysis Consortium (CPTAC) was established to address the need for improved design, standardization, and validation of proteomics assays to enable better translation of biomarkers from the analytical lab to the clinic. Here, we applied CPTAC guidelines to characterize quantitative mass spectrometry (MS) assays in a new multiple reaction monitoring (MRM) proteomics panel. The panel of 50 proteins was developed in response to a previous study that identified a proteomic profile of altered translational control associated with response to a new cancer drug. MRM-MS assays for 53 peptides of interest were developed, optimized, and characterized on a UPLC system coupled to a triple-quadrupole mass spectrometer (QQQ-MS) using synthetic proteotypic peptides and corresponding stable-isotope labeled internal standard (SIS) peptides. Most of the assays were found to be fit-for-purpose for biomarker verification in that they precisely and reproducibly quantify the peptides at levels corresponding to the endogenous concentration in the desired cancer cell lines. Of these, 28 peptide assays represent to proteins that previously had no associated assays published in the CPTAC database. The targeted proteins in this publicly deposited validated multiplexed panel may be of use for research applications in cancer, cellular stress, neurology, cardiology, and metabolism.

As part of the intestinal microbiota, Candida albicans can elicit a humoral response in the gastrointestinal tract (GIT) that is mainly directed toward hyphal antigens. This response has been implicated in controlling the invasive form of the fungus and maintaining the yeast as an innocuous commensal. However, the specific targets of this response are still unknown. Here, we used a gel-free immunoproteomic methodology to identify C. albicans gut immunogens. For this goal, we previously obtained specific secreted IgA from mice colonized with C. albicans. Then, secretome and surfome from C. albicans wild-type filaments were obtained and incubated with magnetic beads linked to antimouse IgA antibodies. sIgA targets were immunoprecipitated and analyzed by mass spectrometry. A third sample bearing the C. albicans antigen–sIgA complex was also examined. Those identified proteins that exhibited a higher percentile of relative abundance were considered for further analysis. From those, 35 proteins coincided among the three samples. Remarkably, about 40% of the identified proteins appeared in the databases as uncharacterized. The results were then validated by immunofluorescence assays overexpressing some of the genes identified in a yeast-lock C. albicans mutant. Adhesins such as Als3, Als1, and Hwp1 were corroborated to be IgA targets, as well as the chaperone Ssa2. Therefore, this gel-free immunoproteomic approach has been useful to identify new C. albicans antigens that generate a specific humoral response in the murine gut.

Variation in parenting behavior is widespread across the animal kingdom, both within and between species. There are two ecotypes of the three-spined stickleback fish (Gasterosteus aculeatus) that exhibit dramatic differences in their paternal behavior. Males of the common ecotype are highly attentive fathers, tending to young from eggs to fry, while males of the white ecotype desert offspring as eggs. As the pituitary is a key regulator in the hypothalamic-pituitary-adrenal (HPA) axis and the hypothalamic-pituitary-gonadal (HPG) axis between the brain and body, its peptides may influence parenting behaviors. Here, we utilized matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) for high-throughput peptide analysis in single cells of pituitaries from both three-spined stickleback ecotypes. Peptide mass fingerprinting was performed using an in silico generated peptide library to identify detected prohormones. Differential analysis revealed POMC-derived peptides, MCH-derived peptides, and oxytocin as significantly different between the two ecotypes, with higher oxytocin levels in the common ecotype. Interestingly, these subtle chemical differences were not captured by Leiden clustering of the cellular phenotypes. These results call for further investigation of the neurochemical basis for parenting in sticklebacks.

Unipept, a pioneering software tool in metaproteomics, has significantly advanced the analysis of complex ecosystems by facilitating both taxonomic and functional insights from environmental samples. From the onset, Unipept’s capabilities focused on tryptic peptides, utilizing the predictability and consistency of trypsin digestion to efficiently construct a protein reference database. However, the evolving landscape of proteomics and emerging fields like immunopeptidomics necessitate a more versatile approach that extends beyond the analysis of tryptic peptides. In this article, we present a significant update to the underlying index structure of Unipept, which is now powered by a Sparse Suffix Array index. This advancement enables the analysis of semitryptic peptides, peptides with missed cleavages, and nontryptic peptides such as those encountered in other research fields such as immunopeptidomics (e.g., MHC- and HLA-peptides). This new index benefits all tools in the Unipept ecosystem such as the web application, desktop tool, application programming interface (API), and command line interface. A benchmark study highlights significantly improved performance in handling missed cleavages, preserving the same level of accuracy.

Pseudomonas aeruginosa (Pa) utilizes heme as an iron source from the host during infection. Biliverdin beta and delta (BVIXβ and BVIXδ) are generated by HemO, specific to Pa, while biliverdin alpha is generated from the bacterial BphO system and by mammalian heme oxygenases. Here, we have developed and characterized a quantitative LC–MS/MS assay for the separation of three endogenous isomers, BVIXα, BVIXβ, and BVIXδ. The assay was validated for accuracy, precision, linearity, extraction recovery, solution stability, freeze–thaw stability, benchtop stability, postextraction stability, and nonspecific oxidation of BVIX. The addition of an antioxidant, butylated hydroxytoluene, during sample preparation is needed in order to prevent coupled oxidation from inflating quantitative values of BVIX. The assay development included optimization of a liquid–liquid extraction for bacterial culture supernatants and sample preparation procedures for cell pellets and tissue homogenate to reduce sample demand and automate the extraction procedure in a 96-well format, to enhance extraction throughput. This method was applied to analyze isomer distribution in Pa supernatant, bacterial pellet, and infected lung tissue from Pa-challenged mice. This method can be used in the future for low-volume culture samples, as well as tissue samples, to understand the mechanisms of virulence and inform future drug development.

Background: Peri-implantitis is characterized as a pathological change in the tissues around dental implants. Fourier-transform infrared spectroscopy (FTIR) provides molecular information from optical phenomena observed by the vibration of molecules, which is used in biological studies to characterize changes and serves as a form of diagnosis. Aims: this case–control study evaluated the peri-implant disease by using FTIR spectroscopy with attenuated total reflectance in the fingerprint region. Methods: 38 saliva samples were evaluated, 17 from the control group and 21 from the peri-implantitis group. Clinical data such as plaque index (PI), gingival index, probing depth (PS), and attachment level were assessed. Results: The results of clinical parameters showed a statistical difference between the two groups regarding an excess of the PI. In the FTIR-ATR analysis, the main components revealed vibrational modes of fatty acids, histidine, lipid esters, nucleic acids, and tryptophan, with the main molecules contributing to spectral discrimination. The five-component partial least-squares discriminant analysis classification model had an accuracy of 81%, showing differences between healthy and diseased implants. Conclusion: the FTIR spectroscopy provides important molecular characteristics of the samples and the results in association with clinical data show the effectiveness of using this tool for diagnosing the disease.

Metabolic reprogramming is important in primary biliary cholangitis (PBC) development. However, studies investigating the metabolic signature within the liver of PBC patients are limited. In this study, liver biopsies from 31 PBC patients and 15 healthy controls were collected, and comprehensive metabolomics, lipidomics, and proteomics analysis were conducted to characterize the metabolic landscape in PBC. We observed distinct lipidome remodeling in PBC with increased polyunsaturated fatty acid levels and augmented fatty acid β-oxidation (FAO), evidenced by the increased acylcarnitine levels and upregulated expression of proteins involved in FAO. Notably, PBC patients exhibited an increase in glucose-6-phosphate (G6P) and purines, alongside a reduction in pyruvate, suggesting impaired glycolysis and increased purines biosynthesis in PBC. Additionally, the accumulation of bile acids as well as a decrease in branched chain amino acids and aromatic amino acids were observed in PBC liver. We also observed an aberrant upregulation of proteins associated with ductular reaction, apoptosis, and autophagy. In conclusion, our study highlighted substantial metabolic reprogramming in glycolysis, fatty acid metabolism, and purine biosynthesis, coupled with aberrant upregulation of proteins associated with apoptosis and autophagy in PBC patients. Targeting the specific metabolic reprogramming may offer potential targets for the therapeutic intervention of PBC.