Proteomic Fingerprint Research Articles

New insights into the pathophysiology of rheumatic valve disease based on different genetic and proteomics profiles

Abstract Introduction Rheumatic heart disease (RHD) pathogenesis is not fully understood. Purpose The protein profile in rheumatic heart disease may have a different fingerprint in the aortic and mitral valves, which results in different evolution in terms of valve damage. Methods To evaluate the genetic profile in symptomatic RHD, 4 simultaneous mitral and aortic valves were obtained during valve replacement, as well as 4 "normal" valves from cadavers (Control Group). The expression of 90 genes was evaluated using a customized RT-PCR array kit. A second experiment was carried out to evaluate proteomics. Then, 7 mitral and aortic valves, from the same patient, were also collected during valve surgery. The Control Group consisted of 6 mitral and aortic valves obtained from explanted hearts during heart transplantation due to non-valvular pathologies. Additionally, histopathological and sectoral proteomic analysis of amyloid deposits, found in some mitral and aortic valves, from the second experiment, were also reported. Results Mitral rheumatic valves exhibited higher expression of genes linked to calcification and immuno-inflammation compared to aortic rheumatic valves (Figure 1). Proteomic analysis, visualized through PCA graphs, highlighted differences in proteomic patterns. Despite having the same genetic pattern, control valves exhibited different proteomic profiles (Figure 1) Rheumatic mitral and aortic valves, even from the same individual, showed distinct proteomic fingerprint (Figure 1). A total of 49 significant proteins were identified in distinguishing healthy mitral and aortic valves from the same individual (Figure 1). The aortic valve displayed a richer extracellular matrix and higher levels of housekeeping proteins that maintain valve homeostasis compared to the mitral valve (Figure 1). The dominant proteomic pattern in valvular rheumatic disease was identified as alterations in critical mitochondrial metabolic activities related to proteostasis. (Figure 2). Particularly notable was the upregulation of pathways associated with lipid metabolism (Figure 2). Finally, amyloid deposits suggested APOA1 as a potential deposit protein in RHD (Figure 2). Conclusion The study confirms distinct constitutional differences between mitral and aortic valves affecting rheumatic disease progression. It emphasizes the role of altered proteostasis and altered proteins deposit, mitochondrial metabolic changes, and inflammatory responses in the disease's pathophysiology, highlighting the complexity of valvular rheumatic disease involvement.For the first time amyloid deposition has been reported as a likely consequence of significant disruption in valvular proteostasis and the proteins potentially comprising this deposit within a rheumatic valve.Genomic and proteomic overviewproteomic fingerprint of RHD and amyloid

Cytotoxicity and mechanisms of perfluorobutane sulfonate (PFBS) in umbilical cord fibroblast cells of Yangtze finless porpoise

Yangtze finless porpoises (YFP) accumulate high levels of per- and polyfluoroalkyl substances (PFASs). However, the health impacts of PFASs to YFP are still unknown because it is technically and ethically unfeasible to use the critically endangered YFP in toxicological exposures. To uncover the potential toxicities of PFASs to YFP, this study exposed a YFP umbilical cord fibroblast cell line to perfluorobutane sulfonate (PFBS), an emerging PFASs pollutant in the aquatic environments. After exposure, the cytotoxicity and mechanisms of PFBS were explored. Our preliminary experiments found that PFBS compromised the cell viability in a concentration and duration dependent manner. In an exposure of 48-h duration, the maximum no observed effect concentration (NOEC) of PFBS was determined to be 400 µM. High-throughput proteomics were then conducted to identify the differentially expressed proteins in YFP cells exposed to 400 µM PFBS for 48 h. The results found that PFBS exposure significantly perturbed the proteome fingerprints of YFP umbilical cord fibroblast cells. Functional annotation of differential proteins showed that PFBS had the potential to impair a variety of biological processes associated with the immunity, oxidative stress, metabolism, and proteolysis. Consistently, the intracellular levels of reactive oxygen species (ROS) and proinflammatory cytokine IL-1β were significantly increased by PFBS in YFP umbilical cord fibroblast cells. Overall, this study highlights the toxic effects of emerging PFASs on YFP and provides reference data to evaluate the health risks of aquatic pollution under the context of national YFP protection. To our knowledge, this is the first omics study using YFP umbilical cord fibroblast cells in ecotoxicology of PFASs, which is applicable to various cetacean species and pollutants.

OCT1 (SLC22A1) transporter kinetics and regulation in primary human hepatocyte 3D spheroids

3D spheroids of primary human hepatocytes (3D PHH) retain a differentiated phenotype with largely conserved metabolic function and proteomic fingerprint over weeks in culture. As a result, 3D PHH are gaining importance as a model for mechanistic liver homeostasis studies and in vitro to in vivo extrapolation (IVIVE) in drug discovery. However, the kinetics and regulation of drug transporters have not yet been assessed in 3D PHH. Here, we used organic cation transporter 1 (OCT1/SLC22A1) as a model to study both transport kinetics and the long-term regulation of transporter activity via relevant signalling pathways. The kinetics of the OCT1 transporter was studied using the fluorescent model substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+) and known OCT1 inhibitors in individual 3D PHH. For long-term studies, 3D PHH were treated with xenobiotics for seven days, after which protein expression and OCT1 function were assessed. Global proteomic analysis was used to track hepatic phenotypes as well as prototypical changes in other regulated proteins, such as P-glycoprotein and Cytochrome P450 3A4. ASP+ kinetics indicated a fully functional OCT1 transporter with a Km value of 14 ± 4.0µM as the mean from three donors. Co-incubation with known OCT1 inhibitors decreased the uptake of ASP+ in the 3D PHH spheroids by 35–52%. The long-term exposure studies showed that OCT1 is relatively stable upon activation of nuclear receptor signalling or exposure to compounds that could induce inflammation, steatosis or liver injury. Our results demonstrate that 3D PHH spheroids express physiologically relevant levels of fully active OCT1 and that its transporter kinetics can be accurately studied in the 3D PHH configuration. We also confirm that OCT1 remains stable and functional during the activation of key metabolic pathways that alter the expression and function of other drug transporters and drug-metabolizing enzymes. These results will expand the range of studies that can be performed using 3D PHH.

Exploratory Mass Spectrometry of Cerebrospinal Fluid from Persons with Autopsy-Confirmed LATE-NC.

Common neuropathologies associated with dementia include Alzheimer's disease neuropathologic change (ADNC) and limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC). Biofluid proteomics provides a window into the pathobiology of dementia and the information from biofluid tests may help guide clinical management. Participants (n = 29) had been autopsied and had antemortem CSF draws in a longitudinal cohort of older adults at the University of Kentucky AD Research Center. Cases were designated as LATE-NC + if they had LATE-NC stage > 1 (n = 9); the remaining 20 cases were designated LATE-NC-. This convenience sample of CSF specimens was analyzed in two separate processes: From one group, aliquots were depleted of highly abundant proteins using affinity spin columns. Tryptic digests of sample proteins were subjected to liquid chromatographic separation and mass spectrometry. Relative quantification was performed using Sciex software. Peptides referent to a total of 949 proteins were identified in the samples depleted of abundant proteins, and 820 different proteins were identified in the non-depleted samples. When the Bonferroni/false-discovery statistical correction was applied to account for having made multiple comparison tests, only 4 proteins showed differential expression (LATE-NC + vs LATE-NC-) in the non-depleted samples (RBP4, MIF, IGHG3, and ITM2B). Post hoc western blots confirmed that RBP4 expression was higher in the LATE-NC + cases at the group level. In summary, an exploratory assessment of proteomes of autopsy-confirmed LATE-NC and non-LATE-NC CSF did not demonstrate a clear-cut proteomic fingerprint that distinguished the two groups. There was, however, an increase in RBP4 protein levels in CSF from LATE-NC cases.

Very short sleep duration reveals a proteomic fingerprint that is selectively associated with incident diabetes mellitus but not with incident coronary heart disease: a cohort study

BackgroundThe molecular pathways linking short and long sleep duration with incident diabetes mellitus (iDM) and incident coronary heart disease (iCHD) are not known. We aimed to identify circulating protein patterns associated with sleep duration and test their impact on incident cardiometabolic disease.MethodsWe assessed sleep duration and measured 78 plasma proteins among 3336 participants aged 46–68 years, free from DM and CHD at baseline, and identified cases of iDM and iCHD using national registers. Incident events occurring in the first 3 years of follow-up were excluded from analyses. Tenfold cross-fit partialing-out lasso logistic regression adjusted for age and sex was used to identify proteins that significantly predicted sleep duration quintiles when compared with the referent quintile 3 (Q3). Predictive proteins were weighted and combined into proteomic scores (PS) for sleep duration Q1, Q2, Q4, and Q5. Combinations of PS were included in a linear regression model to identify the best predictors of habitual sleep duration. Cox proportional hazards regression models with sleep duration quintiles and sleep-predictive PS as the main exposures were related to iDM and iCHD after adjustment for known covariates.ResultsSixteen unique proteomic markers, predominantly reflecting inflammation and apoptosis, predicted sleep duration quintiles. The combination of PSQ1 and PSQ5 best predicted sleep duration. Mean follow-up times for iDM (n = 522) and iCHD (n = 411) were 21.8 and 22.4 years, respectively. Compared with sleep duration Q3, all sleep duration quintiles were positively and significantly associated with iDM. Only sleep duration Q1 was positively and significantly associated with iCHD. Inclusion of PSQ1 and PSQ5 abrogated the association between sleep duration Q1 and iDM. Moreover, PSQ1 was significantly associated with iDM (HR = 1.27, 95% CI: 1.06–1.53). PSQ1 and PSQ5 were not associated with iCHD and did not markedly attenuate the association between sleep duration Q1 with iCHD.ConclusionsWe here identify plasma proteomic fingerprints of sleep duration and suggest that PSQ1 could explain the association between very short sleep duration and incident DM.

Predicting Cellular Rejection of Renal Allograft Based on the Serum Proteomic Fingerprint.

Kidney transplantation is an essential medical procedure that significantly enhances the survival rates and quality of life for patients with end-stage kidney disease. However, despite advancements in immunosuppressive therapies, allograft rejection remains a leading cause of organ loss. Notably, predictions of cellular rejection processes primarily rely on biopsy analysis, which is not routinely performed due to its invasive nature. The present work evaluates if the serum proteomic fingerprint, as acquired by Fourier Transform Infrared (FTIR) spectroscopy, can predict cellular rejection processes. We analyzed 28 serum samples, corresponding to 17 without cellular rejection processes and 11 associated with cellular rejection processes, as based on biopsy analyses. The leave-one-out-cross validation procedure of a Naïve Bayes model enabled the prediction of cellular rejection processes with high sensitivity and specificity (AUC > 0.984). The serum proteomic profile was obtained in a high-throughput mode and based on a simple, rapid, and economical procedure, making it suitable for routine analyses and large-scale studies. Consequently, the current method presents a high potential to predict cellular rejection processes translatable to clinical scenarios, and that should continue to be explored.

A universal tool for marine metazoan species identification: towards best practices in proteomic fingerprinting

Proteomic fingerprinting using MALDI-TOF mass spectrometry is a well-established tool for identifying microorganisms and has shown promising results for identification of animal species, particularly disease vectors and marine organisms. And thus can be a vital tool for biodiversity assessments in ecological studies. However, few studies have tested species identification across different orders and classes. In this study, we collected data from 1246 specimens and 198 species to test species identification in a diverse dataset. We also evaluated different specimen preparation and data processing approaches for machine learning and developed a workflow to optimize classification using random forest. Our results showed high success rates of over 90%, but we also found that the size of the reference library affects classification error. Additionally, we demonstrated the ability of the method to differentiate marine cryptic-species complexes and to distinguish sexes within species.

The proteomic landscape of sperm surface deciphers its maturational and functional aspects in buffalo.

Buffalo is a dominant dairy animal in many agriculture-based economies. However, the poor reproductive efficiency (low conception rate) of the buffalo bulls constrains the realization of its full production potential. This in turn leads to economic and welfare issues, especially for the marginal farmers in such economies. The mammalian sperm surface proteins have been implicated in the regulation of survival and function of the spermatozoa in the female reproductive tract (FRT). Nonetheless, the lack of specific studies on buffalo sperm surface makes it difficult for researchers to explore and investigate the role of these proteins in the regulation of mechanisms associated with sperm protection, survival, and function. This study aimed to generate a buffalo sperm surface-specific proteomic fingerprint (LC-MS/MS) and to predict the functional roles of the identified proteins. The three treatments used to remove sperm surface protein viz. Elevated salt, phosphoinositide phospholipase C (PI-PLC) and in vitro capacitation led to the identification of N = 1,695 proteins (≥1 high-quality peptide-spectrum matches (PSMs), p < 0.05, and FDR<0.01). Almost half of these proteins (N = 873) were found to be involved in crucial processes relevant in the context of male fertility, e.g., spermatogenesis, sperm maturation and protection in the FRT, and gamete interaction or fertilization, amongst others. The extensive sperm-surface proteomic repertoire discovered in this study is unparalleled vis-à-vis the depth of identification of reproduction-specific cell-surface proteins and can provide a potential framework for further studies on the functional aspects of buffalo spermatozoa.

Transcriptomic and proteomic fingerprints induced by the fungicides difenoconazole and metalaxyl in zebrafish embryos

In this study, we applied OMICs analysis to identify substance-specific biomarker candidates, which may act as early indicators for specific ecotoxic modes of actions (MoA). Zebrafish embryos were exposed to two sublethal concentrations of difenoconazole and metalaxyl according to a modified protocol of the OECD test guideline No. 236. At the end of exposure, total RNA and protein were extracted, followed by transcriptomics and proteomics analysis. The analysis of significantly differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) revealed a positive exposure-response correlation in all test concentrations for both fungicides. Similarly, also a positive correlation between the obtained transcriptome and proteome data was observed, highlighting the robustness of our approach. From the detected DEGs, candidate biomarkers specific for difenoconazole (apoa1b, gatm, mylpfb and acta1b) and metalaxyl (lgals2b, abat, fabp1b.1 and myh9a) were selected, and their biological functions were discussed to assess the predictive potential.

The proteomic fingerprint in infants with single ventricle heart disease in the interstage period: evidence of chronic inflammation and widespread activation of biological networks.

Children with single ventricle heart disease (SVHD) experience significant morbidity across systems and time, with 70% of patients experiencing acute kidney injury, 33% neurodevelopmental impairment, 14% growth failure, and 5.5% of patients suffering necrotizing enterocolitis. Proteomics is a method to identify new biomarkers and mechanisms of injury in complex physiologic states. Infants with SVHD in the interstage period were compared to similar-age healthy controls. Serum samples were collected, stored at -80°C, and run on a panel of 1,500 proteins in single batch analysis (Somalogic Inc., CO). Partial Least Squares-Discriminant Analysis (PLS-DA) was used to compare the proteomic profile of cases and controls and t-tests to detect differences in individual proteins (FDR <0.05). Protein network analysis with functional enrichment was performed in STRING and Cytoscape. PLS-DA readily discriminated between SVHD cases (n = 33) and controls (n = 24) based on their proteomic pattern alone (Accuracy = 0.96, R2 = 0.97, Q2 = 0.80). 568 proteins differed between groups (FDR <0.05). We identified 25 up-regulated functional clusters and 13 down-regulated. Active biological systems fell into six key groups: angiogenesis and cell proliferation/turnover, immune system activation and inflammation, altered metabolism, neural development, gastrointestinal system, and cardiac physiology and development. We report a clear differentiation in the circulating proteome of patients with SVHD and healthy controls with >500 circulating proteins distinguishing the groups. These proteomic data identify widespread protein dysregulation across multiple biologic systems with promising biological plausibility as drivers of SVHD morbidity.

Abstract 17103: The Proteomic Fingerprint in Patients With Single Ventricle Disease in the Interstage Period: Evidence of Chronic Inflammation and Widespread Activation of Biological Networks

Introduction: Children with single ventricle heart disease (SVHD) experience significant morbidity across systems and time: (AKI 70%, neurodevelopmental impairment 33%, growth failure 14%, NEC 5.5%). Proteomics is a novel method to identify new biomarkers and mechanisms of injury in complex physiologic states. Hypothesis: Does the proteome of patients with SVHD differ from healthy children, and which biological systems are activated or repressed? Methods: Infants with SVHD in the interstage period were compared to similar-age healthy controls. Serum samples were collected, stored at -80C, and run on a panel of 1500 proteins in single batch analysis (Somalogic Inc., CO). Partial Least Squares-Discriminant Analysis (PLS-DA) was used to compare the proteomic profile of cases and controls and t-tests to detect differences in individual proteins (FDR &lt; 0.05). Protein network analysis with functional enrichment was performed in STRING and Cytoscape. Results: PLS-DA readily discriminated between cases (n=33) and controls (n=24) based on their proteomic pattern alone (Accuracy=0.96, R2=0.97, Q2=0.80) (Fig 1a). 568 proteins differed between groups (FDR &lt; 0.05). The 15 proteins with the greatest effect on the proteomic phenotype between the groups are shown (Fig 1b). We identified 25 up-regulated functional clusters and 13 down-regulated (5 clusters highlighted in Fig 1c). Active biological systems fell into six key groups: angiogenesis and cell proliferation/turnover, immune system activation and inflammation, altered metabolism, neuronal development, gastrointestinal system, and cardiac development. Conclusions: We report a clear differentiation in the circulating proteome of patients with SVHD and healthy controls with &gt;500 circulating proteins distinguishing the groups. This proteomic data identifies various dysregulated protein networks across multiple organ systems with promising biological plausibility as drivers of SVHD morbidity.

Preomic profile of BxPC-3 cells after treatment with BRC4

BRCA2 and RAD51 are two proteins that play a central role in homologous recombination (HR) and DNA double strand break (DSB) repair. BRCA2 assists RAD51 fibrillation and defibrillation through binding with its eight BRC repeats, with BRC4 being one of the most efficient and best characterized. RAD51 inactivation by small molecules has been proposed as a strategy to impair BRCA2/RAD51 binding and, ultimately, the HR pathway, with the aim of making cancer cells more sensitive to PARP inhibitors (PARPi). This strategy, which mimics a synthetic lethality (SL) approach, has been successfully performed in vitro by using the myristoylated derivative of BRC4 (myr-BRC4), designed for a more efficient cell entry. The present study applies a method to obtain a proteomic fingerprint after cellular treatment with the myr-BRC4 peptide using a mass spectroscopy (MS) proteomic approach. (Data are available via ProteomeXchange with identifier PXD042696.) We performed a comparative proteomic profiling of the myr-BRC4 treated vs. untreated BxPC-3 pancreatic cancer cells and evaluated the differential expression of proteins. Among the identified proteins, we focused our attention on proteins shared by both the RAD51 and the BRCA2 interactomes, and on those whose reduction showed high statistical significance. Three downregulated proteins were identified (FANCI, FANCD2, and RPA3), and protein downregulation was confirmed through immunoblotting analysis, validating the MS approach. Our results suggest that, being a direct consequence of myr-BRC4 treatment, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring HR impairment. SignificanceRAD51's inhibition has gained increasing attention because of its possible implications in personalized medicine through the SL approach. Chemical disruption of protein-protein interactions (PPIs) between RAD51 and BRCA2, or some of its partner proteins, could potentiate PARPi DNA damage-induced cell death. This could have application for difficult to treat cancers, such as BRCA-competent and olaparib (PARPi) resistant pancreatic adenocarcinoma. Despite RAD51 being a widely studied target, researchers still lack detailed mechanistic information. This has stifled progress in the field with only a few RAD51 inhibitors having been identified, none of which have gained regulatory approval. Nevertheless, the peptide BRC4 is one of the most specific and best characterized RAD51 binder and inhibitor reported to date. Our study is the first to report the proteomic fingerprint consequent to cellular treatment of myr-BRC4, to offer a reference for the discovery of specific protein/pathway alterations within DNA damage repair. Our results suggest that, being a direct consequence of myr-BRC4 treatment, and ultimately ofBRCA2/RAD51 disruption, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring DNA damage repair impairment and therefore be used to potentiate the development of new effective therapeutic strategies.

Potential of MALDI-TOF MS-based proteomic fingerprinting for species identification of Cnidaria across classes, species, regions and developmental stages.

Morphological identification of cnidarian species can be difficult throughout all life stages due to the lack of distinct morphological characters. Moreover, in some cnidarian taxa genetic markers are not fully informative, and in these cases combinations of different markers or additional morphological verifications may be required. Proteomic fingerprinting based on MALDI-TOF mass spectra was previously shown to provide reliable species identification in different metazoans including some cnidarian taxa. For the first time, we tested the method across four cnidarian classes (Staurozoa, Scyphozoa, Anthozoa, Hydrozoa) and included different scyphozoan life-history stages (polyp, ephyra, medusa) in our dataset. Our results revealed reliable species identification based on MALDI-TOF mass spectra across all taxa with species-specific clusters for all 23 analysed species. In addition, proteomic fingerprinting was successful for distinguishing developmental stages, still by retaining a species specific signal. Furthermore, we identified the impact of different salinities in different regions (North Sea and Baltic Sea) on proteomic fingerprints to be negligible. In conclusion, the effects of environmental factors and developmental stages on proteomic fingerprints seem to be low in cnidarians. This would allow using reference libraries built up entirely of adult or cultured cnidarian specimens for the identification of their juvenile stages or specimens from different geographic regions in future biodiversity assessment studies.

How eluents define proteomic fingerprinting of protein corona on nanoparticles

Nanoparticles (NPs) have broad application prospects in the field of biomedicine due to their excellent physicochemical properties. When entering biological fluids, NPs inevitably encountered proteins and were subsequently surrounded by them, forming the termed protein corona (PC). As PC has been evidenced to have critical roles in deciding the biological fates of NPs, how to precisely characterize PC is vital to promote the clinical translation of nanomedicine by understanding and harnessing NPs’ behaviors. During the centrifugation-based separation techniques for the PC preparation, direct elution has been most widely used to strip proteins from NPs due to its simpleness and robustness, but the roles of multifarious eluents have never been systematically declared. Herein, seven eluents composed of three denaturants, sodium dodecyl sulfate (SDS), dithiothreitol (DTT), and urea (Urea), were applied to detach PC from gold nanoparticles (AuNPs) and silica nanoparticles (SiNPs), and eluted proteins in PC have been carefully characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and chromatography coupled tandem mass spectrometry (LC-MS/MS). Our results showed that SDS and DTT were the main contributors to the efficient desorption of PC on SiNPs and AuNPs, respectively. The molecular reactions between NPs and proteins were explored and verified by SDS-PAGE analysis of PC formed in the serums pretreated with protein denaturing or alkylating agents. The proteomic fingerprinting analysis indicated the difference of the eluted proteins brought by the seven eluents was the abundance rather than the species. The enrichment of some opsonins and dysopsonins in a special elution reminds us that the possibility of biased judgments on predicting NPs’ biological behaviors under different elution conditions. The synergistic effects or antagonistic effects among denaturants for eluting PC were manifested in a nanoparticle-type dependent way by integrating the properties of the eluted proteins. Collectively, this study not only underlines the urgent need of choosing the appropriate eluents for identifying PC robustly and unbiasedly, but also provides an insight into the understanding of molecular interactions during PC formation.

Cyanide Biodegradation by a Native Bacterial Consortium and Its Potential for Goldmine Tailing Biotreatment

A native cyanide-degrading bacterial consortium was isolated from goldmine tailing sediments. Mine tailings are toxic effluents due to their metal–cyanide complexes. The bacterial consortium was able to degrade an initial sodium cyanide concentration ranging from 5 to 120 mg L−1 in alkaline synthetic wastewater (pH &gt; 9.2), for a maximum of 15 days. The free cyanide biodegradation efficiency was 98% for the highest initial free cyanide concentration tested and followed a first-order kinetic profile, with an estimated kinetic rate constant of 0.12 ± 0.011 d−1. The cyanide-degrading consortium was streaked with serial dilutions on a specific medium (R2A). 16S rRNA gene sequencing and mass spectrometry proteomic fingerprinting of the isolates showed that the bacterial strains belonged to Microbacterium paraoxydans, Brevibacterium casei, Brevundimonas vesicularis, Bacillus cereus and Cellulosimicrobium sp. The first four genera had previously been identified as cyanide-degrading bacteria. Microbacterium and Brevibacterium had previously been found in alkaline conditions, showing resistance to heavy metals. As for Cellulosimicrobium, to our knowledge, this is the first study to implicate it directly or indirectly in cyanide biodegradation. In this research, these genera were identified as functional bacteria for cyanide degradation, and they might be suitable for mine tailing biotechnological tertiary treatment.

Robust Acute Pancreatitis Identification and Diagnosis: RAPIDx

The occurrence of acute pancreatitis (AP) is increasing significantly worldwide. However, current diagnostic methods of AP do not provide a clear clinical stratification of severity, and the prediction of complications in AP is still limited. Here, we present a robust AP identification and diagnosis (RAPIDx) method by the proteomic fingerprinting of intact nanoscale extracellular vesicles (EVs) from clinical samples. By tracking analysis of circulating biological nanoparticles released by cells (i.e., EVs) via bottom-up proteomics, we obtain close phenotype connections between EVs, cell types, and multiple tissues based on their specific proteomes and identify the serum amyloid A (SAA) proteins on EVs as potential biomarkers that are differentially expressed from AP patients significantly. We accomplish the quantitative analysis of EVs fingerprints using MALDI-TOF MS and find the SAA proteins (SAA1-1, desR-SAA1-2, SAA2, SAA1-2) with areas under the curve (AUCs) from 0.92 to 0.97, which allows us to detect AP within 30 min. We further realize that SAA1-1 and SAA2, combined with two protein peaks (5290.19, 14032.33 m/z), can achieve an AUC of 0.83 for classifying the severity of AP. The RAPIDx platform will facilitate timely diagnosis and treatment of AP before severity development and persistent organ failure and promote precision diagnostics and the early diagnosis of pancreatic cancer.

Perspectives of species identification by MALDI-TOF MS in monitoring-Stability of proteomic fingerprints in marine epipelagic copepods.

We analysed the robustness of species identification based on proteomic composition to data processing and intraspecific variability, specificity and sensitivity of species-markers as well as discriminatory power of proteomic fingerprinting and its sensitivity to phylogenetic distance. Our analysis is based on MALDI-TOF MS (matrix-assisted laser desorption ionization time of flight mass spectrometry) data from 32 marine copepod species coming from 13 regions (North and Central Atlantic and adjacent seas). A random forest (RF) model correctly classified all specimens to the species level with only small sensitivity to data processing, demonstrating the strong robustness of the method. Compounds with high specificity showed low sensitivity, that is identification was based on complex pattern-differences rather than on presence of single markers. Proteomic distance was not consistently related to phylogenetic distance. A species-gap in proteome composition appeared at 0.7 Euclidean distance when using only specimens from the same sample. When other regions or seasons were included, intraspecific variability increased, resulting in overlaps of intra and inter-specific distance. Highest intraspecific distances (>0.7) were observed between specimens from brackish and marine habitats (i.e., salinity probably affects proteomic patterns). When testing library sensitivity of the RF model to regionality, strong misidentification was only detected between two congener pairs. Still, the choice of reference library may have an impact on identification of closely related species and should be tested before routine application. We envisage high relevance of this time- and cost-efficient method for future zooplankton monitoring as it provides not only in-depth taxonomic resolution for counted specimens but also add-on information, such as on developmental stage or environmental conditions.

Label-free single cell proteomics utilizing ultrafast LC and MS instrumentation: A valuable complementary technique to multiplexing.

The ability to map a proteomic fingerprint to transcriptomic data would master the understanding of how gene expression translates into actual phenotype. In contrast to nucleic acid sequencing, in vitro protein amplification is impossible and no single cell proteomic workflow has been established as gold standard yet. Advances in microfluidic sample preparation, multi-dimensional sample separation, sophisticated data acquisition strategies, and intelligent data analysis algorithms have resulted in major improvements to successfully analyze such tiny sample amounts with steadily boosted performance. However, among the broad variation of published approaches, it is commonly accepted that highest possible sensitivity, robustness, and throughput are still the most urgent needs for the field. While many labs have focused on multiplexing to achieve these goals, label-free SCP is a highly promising strategy as well whenever high dynamic range and unbiased accurate quantification are needed. We here focus on recent advances in label-free single-cell mass spectrometry workflows and try to guide our readers to choose the best method or combinations of methods for their specific applications. We further highlight which techniques are most propitious in the future and which applications but also limitations we foresee for the field.

Proteomic Fingerprint of Lung Fibrosis Progression and Response to Therapy in Bleomycin-Induced Mouse Model.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by the aberrant accumulation of extracellular matrix in the lungs. nintedanib is one of the two FDA-approved drugs for IPF treatment; however, the exact pathophysiological mechanisms of fibrosis progression and response to therapy are still poorly understood. In this work, the molecular fingerprint of fibrosis progression and response to nintedanib treatment have been investigated by mass spectrometry-based bottom-up proteomics in paraffin-embedded lung tissues from bleomycin-induced (BLM) pulmonary fibrosis mice. Our proteomics results unveiled that (i) samples clustered depending on the tissue fibrotic grade (mild, moderate, and severe) and not on the time course after BLM treatment; (ii) the dysregulation of different pathways involved in fibrosis progression such as the complement coagulation cascades, advanced glycation end products (AGEs) and their receptors (RAGEs) signaling, the extracellular matrix-receptor interaction, the regulation of actin cytoskeleton, and ribosomes; (iii) Coronin 1A (Coro1a) as the protein with the highest correlation when evaluating the progression of fibrosis, with an increased expression from mild to severe fibrosis; and (iv) a total of 10 differentially expressed proteins (padj-value ≤ 0.05 and Fold change ≤-1.5 or ≥1.5), whose abundance varied in the base of the severity of fibrosis (mild and moderate), were modulated by the antifibrotic treatment with nintedanib, reverting their trend. Notably, nintedanib significantly restored lactate dehydrogenase B (Ldhb) expression but not lactate dehydrogenase A (Ldha). Notwithstanding the need for further investigations to validate the roles of both Coro1a and Ldhb, our findings provide an extensive proteomic characterization with a strong relationship with histomorphometric measurements. These results unveil some biological processes in pulmonary fibrosis and drug-mediated fibrosis therapy.

Evaluating species richness using proteomic fingerprinting and DNA barcoding—a case study on meiobenthic copepods from the Clarion Clipperton Fracture Zone

The Clarion Clipperton Fracture Zone (CCZ) is a vast deep-sea region harboring a highly diverse benthic fauna, which will be affected by potential future deep-sea mining of metal-rich polymetallic nodules. Despite the need for conservation plans and monitoring strategies in this context, the majority of taxonomic groups remain scientifically undescribed. However, molecular rapid assessment methods such as DNA barcoding and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) provide the potential to accelerate specimen identification and biodiversity assessment significantly in the deep-sea areas. In this study, we successfully applied both methods to investigate the diversity of meiobenthic copepods in the eastern CCZ, including the first application of MALDI-TOF MS for the identification of these deep-sea organisms. Comparing several different species delimitation tools for both datasets, we found that biodiversity values were very similar, with Pielou’s evenness varying between 0.97 and 0.99 in all datasets. Still, direct comparisons of species clusters revealed differences between all techniques and methods, which are likely caused by the high number of rare species being represented by only one specimen, despite our extensive dataset of more than 2000 specimens. Hence, we regard our study as a first approach toward setting up a reference library for mass spectrometry data of the CCZ in combination with DNA barcodes. We conclude that proteome fingerprinting, as well as the more established DNA barcoding, can be seen as a valuable tool for rapid biodiversity assessments in the future, even when no reference information is available.