Bats are increasingly studied as model systems for longevity and as natural hosts for some virulent viruses. Yet the ability to characterize immune mechanisms of viral tolerance and to quantify infection dynamics in wild bats is often limited by small sample volumes and few species-specific reagents. Here, we demonstrate how proteomics can overcome these limitations by using data-independent acquisition-based shotgun proteomics to survey the serum proteome of 17 vampire bats (Desmodus rotundus) from Belize. Using just 2 μL of sample and relatively short separations of undepleted serum digests, we identified 361 proteins across 5 orders of magnitude. Levels of immunological proteins in vampire bat serum were then compared to human plasma via published databases. Of particular interest were antiviral and antibacterial components, circulating 20S proteasome complex and proteins involved in redox activity. Lastly, we used known virus proteomes to putatively identify Rh186 from Macacine herpesvirus 3 and ORF1a from Middle East respiratory syndrome-related coronavirus, indicating that mass spectrometry-based techniques show promise for pathogen detection. Overall, these results can be used to design targeted mass-spectrometry assays to quantify immunological markers and detect pathogens. More broadly, our findings also highlight the application of proteomics in advancing wildlife immunology and pathogen surveillance.

Update of medRxiv. 2020 Nov 04;:

Immunoglobulin G (IgG) glycosylation is studied in biological samples to develop clinical markers for precision medicine, for example, in autoimmune diseases and oncology. Inappropriate storage of proteins, lipids, or metabolites can lead to degradation or modification of biomolecular features, which can have a strong negative impact on accuracy and precision of clinical omics studies. Regarding the preservation of IgG glycosylation, the range of appropriate storage conditions and time frame is understudied. Therefore, we investigated the effect of storage on IgG Fc N-glycosylation in the commonly analyzed biofluids, serum and plasma. Short-term storage and accelerated storage stability were tested by incubating samples from three healthy donors under stress conditions of up to 50 °C for 2 weeks using -80 °C for 2 weeks as the reference condition. All tested IgG glycosylation features-sialylation, galactosylation, bisection, and fucosylation-remained unchanged up to room temperature as well as during multiple freeze-thaw cycles and exposure to light. Only when subjected to 37 °C or 50 °C for 2 weeks, galactosylation and sialylation subtly changed. Therefore, clinical IgG glycosylation analysis does not rely as heavily on mild serum and plasma storage conditions and timely analysis as many other omics analyses.

The impact of numerous diseases has been linked to differences in sex between organisms, including various neurological diseases. As neuropeptides are known to be key players in the nervous system, studying the variation of neuropeptidomic profiles between males and females in a crustacean model organism is of interest. By using high-resolution mass spectrometry with two complementary ionization sources in conjunction with quantitative chemical labeling (isotopic reductive dimethylation), differences were observed in five key neural tissues and hemolymph. Interestingly, while males and females possess numerous neuropeptide isoforms that are unique to their sex, the represented families of each sex remain largely consistent. However, some differences in familial isoforms were also observed, such as the relative numbers of neuropeptides belonging to RFamide and allatostatin A-type families. Additionally, >100 neuropeptides detected across five neural tissues and hemolymph were found to have statistically significant differences in abundance between male and female blue crab samples. Also, hundreds of putative peptide sequences were identified by de novo sequencing that may be indicative of previously undiscovered neuropeptides, highlighting the power of using a multifaceted MS approach.

Proteomic investigations of Alzheimer's and Parkinson's disease have provided valuable insights into neurodegenerative disorders. Thus far, these investigations have largely been restricted to bottom-up approaches, hindering the degree to which one can characterize a protein's "intact" state. Top-down proteomics (TDP) overcomes this limitation; however, it is typically limited to observing only the most abundant proteoforms and of a relatively small size. Therefore, fractionation techniques are commonly used to reduce sample complexity. Here, we investigate gas-phase fractionation through high-field asymmetric waveform ion mobility spectrometry (FAIMS) within TDP. Utilizing a high complexity sample derived from Alzheimer's disease (AD) brain tissue, we describe how the addition of FAIMS to TDP can robustly improve the depth of proteome coverage. For example, implementation of FAIMS with external compensation voltage (CV) stepping at -50, -40, and -30 CV could more than double the mean number of non-redundant proteoforms, genes, and proteome sequence coverage compared to without FAIMS. We also found that FAIMS can influence the transmission of proteoforms and their charge envelopes based on their size. Importantly, FAIMS enabled the identification of intact amyloid beta (Aβ) proteoforms, including the aggregation-prone Aβ1-42 variant which is strongly linked to AD. Raw data and associated files have been deposited to the ProteomeXchange Consortium via the MassIVE data repository with data set identifier PXD023607.

Proteogenomic approaches have enabled the generat̲ion of novel information levels when compared to single omics studies although burdened by extensive experimental efforts. Here, we improved a data-independent acquisition mass spectrometry proteogenomic workflow to reveal distinct molecular features related to mammographic appearances in breast cancer. Our results reveal splicing processes detectable at the protein level and highlight quantitation and pathway complementarity between RNA and protein data. Furthermore, we confirm previously detected enrichments of molecular pathways associated with estrogen receptor-dependent activity and provide novel evidence of epithelial-to-mesenchymal activity in mammography-detected spiculated tumors. Several transcript-protein pairs displayed radically different abundances depending on the overall clinical properties of the tumor. These results demonstrate that there are differentially regulated protein networks in clinically relevant tumor subgroups, which in turn alter both cancer biology and the abundance of biomarker candidates and drug targets.

Cross-linking mass spectrometry (XL-MS) has become a powerful technique that enables insights into protein structures and protein interactions. The development of cleavable cross-linkers has further promoted XL-MS through search space reduction, thereby allowing for proteome-wide studies. These new analysis possibilities foster the development of new cross-linkers, which not every search engine can deal with out of the box. In addition, some search engines for XL-MS data also struggle with the validation of identified cross-linked peptides, that is, false discovery rate (FDR) estimation, as FDR calculation is hampered by the fact that not only one but two peptides in a single spectrum have to be correct. We here present our new search engine, MS Annika, which can identify cross-linked peptides in MS2 spectra from a wide variety of cleavable cross-linkers. We show that MS Annika provides realistic estimates of FDRs without the need of arbitrary score cutoffs, being able to provide on average 44% more identifications at a similar or better true FDR than comparable tools. In addition, MS Annika can be used on proteome-wide studies due to fast, parallelized processing and provides a way to visualize the identified cross-links in protein 3D structures.

Nutrient-dense dairy foods are an important component of a healthy diet. Recommendations, however, advise non- and low-fat dairy foods despite controversy concerning whether full-fat dairy foods adversely impact cardiometabolic health. Therefore, in this study, our objective is to examine the differential plasma lipidomic responses to non-fat or full-fat milk ingestion during postprandial hyperglycemia. Seven adults with prediabetes completed a randomized cross-over study in which glucose was consumed alone or with non-fat or full-fat dairy milk. Plasma samples collected at 90 min and 180 min post milk ingestion were used to perform untargeted lipidomics analysis. A total of 332 lipids from 20 classes and five lipid categories were detected at different time points during the postprandial period. Dairy milk, especially non-fat milk, protected against lipid changes otherwise induced by glucose ingestion. Co-ingestion of dairy milk with glucose, regardless of fat content, significantly altered lipid profiles although full-fat milk more substantially modulated lipid profiles. For the identified lipid biomarkers, 68.0% and 66.7% of the lipids significantly increased at 90 and 180 min, respectively, while phosphatidylcholines (GPs) contributed most for the significant increase. Comparative lipidomics analysis indicated that both types of dairy milk induced significant changes in several lipid pathways, including glycerophospholipid metabolism and α-linolenic acid metabolism, to protect against postprandial hyperglycemia. In summary, our comparative lipidomics results suggested that dairy milk-mediated lipid modulation may be an effective dietary approach to reduce the risk of metabolic diseases among those with prediabetes.

The outbreak of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed a serious threat to global public health. The mechanism of pathogenesis and the host immune response to SARS-CoV-2 infection are largely unknown. In the present study, we applied a quantitative proteomic technology to identify and quantify the ubiquitination changes that occur in both the virus and the Vero E6 cells during SARS-CoV-2 infection. By applying label-free, quantitative liquid chromatography with tandem mass spectrometry proteomics, 8943 lysine ubiquitination sites on 3086 proteins were identified, of which 138 sites on 104 proteins were quantified as significantly upregulated, while 828 sites on 447 proteins were downregulated at 72 h post-infection. Bioinformatics analysis suggested that SARS-CoV-2 infection might modulate host immune responses through the ubiquitination of important proteins, including USP5, IQGAP1, TRIM28, and Hsp90. Ubiquitination modification was also observed on 11 SAR-CoV-2 proteins, including proteins involved in virus replication and inhibition of the host innate immune response. Our study provides new insights into the interaction between SARS-CoV-2 and the host as well as potential targets for the prevention and treatment of COVID-19.

Moving from macroscale preparative systems in proteomics to micro- and nanotechnologies offers researchers the ability to deeply profile smaller numbers of cells that are more likely to be encountered in clinical settings. Herein a recently developed microscale proteomic method, microdroplet processing in one pot for trace samples (microPOTS), was employed to identify proteomic changes in ∼200 Barrett's esophageal cells following physiologic and radiation stress exposure. From this small population of cells, microPOTS confidently identified >1500 protein groups, and achieved a high reproducibility with a Pearson's correlation coefficient value of R > 0.9 and over 50% protein overlap from replicates. A Barrett's cell line model treated with either lithocholic acid (LCA) or X-ray had 21 (e.g., ASNS, RALY, FAM120A, UBE2M, IDH1, ESD) and 32 (e.g., GLUL, CALU, SH3BGRL3, S100A9, FKBP3, AGR2) overexpressed proteins, respectively, compared to the untreated set. These results demonstrate the ability of microPOTS to routinely identify and quantify differentially expressed proteins from limited numbers of cells.

Misfolding and aggregation of amyloid-β peptide and hyperphosphorylated tau are molecular markers of Alzheimer's disease (AD), and although the 3D structures of these aberrantly folded proteins have been visualized in exquisite detail, no method has been able to survey protein folding across the proteome in AD. Here, we present covalent protein painting (CPP), a mass spectrometry-based protein footprinting approach to quantify the accessibility of lysine ε-amines for covalent modification at the surface of natively folded proteins. We used CPP to survey the reactivity of 2645 lysine residues and therewith the structural proteome of HEK293T cells and found that reactivity increased upon mild heat shock. CPP revealed that the accessibility of lysine residues for covalent modification in tubulin-β (TUBB), in succinate dehydrogenase (SHDB), and in amyloid-β peptide (Aβ) is altered in human postmortem brain samples of patients with neurodegenerative diseases. The structural alterations of TUBB and SHDB in patients with AD, dementia with Lewy bodies (DLB), or both point to broader perturbations of the 3D proteome beyond Aβ and hyperphosphorylated tau.

Osteoarthritis (OA) is a multifactorial pathology and comprises a wide range of distinct phenotypes. In this context, the characterization of the different molecular profiles associated with each phenotype can improve the classification of OA. In particular, OA can coexist with type 2 diabetes mellitus (T2DM). This study investigates lipidomic and proteomic differences between human OA/T2DM- and OA/T2DM+ cartilage through a multimodal mass spectrometry approach. Human cartilage samples were obtained after total knee replacement from OA/T2DM- and OA/T2DM+ patients. Label-free proteomics was employed to study differences in protein abundance and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) for spatially resolved-lipid analysis. Label-free proteomic analysis showed differences between OA/T2DM- and OA/T2DM+ phenotypes in several metabolic pathways such as lipid regulation. Interestingly, phospholipase A2 protein was found increased within the OA/T2DM+ cohort. In addition, MALDI-MSI experiments revealed that phosphatidylcholine and sphingomyelin species were characteristic of the OA/T2DM- group, whereas lysolipids were more characteristic of the OA/T2DM+ phenotype. The data also pointed out differences in phospholipid content between superficial and deep layers of the cartilage. Our study shows distinctively different lipid and protein profiles between OA/T2DM- and OA/T2DM+ human cartilage, demonstrating the importance of subclassification of the OA disease for better personalized treatments.

There is an urgent need to elucidate the underlying mechanisms of coronavirus disease (COVID-19) so that vaccines and treatments can be devised. Severe acute respiratory syndrome coronavirus 2 has genetic similarity with bats and pangolin viruses, but a comprehensive understanding of the functions of its proteins at the amino acid sequence level is lacking. A total of 4320 sequences of human and nonhuman coronaviruses was retrieved from the Global Initiative on Sharing All Influenza Data and the National Center for Biotechnology Information. This work proposes an optimization method COVID-Pred with an efficient feature selection algorithm to classify the species-specific coronaviruses based on physicochemical properties (PCPs) of their sequences. COVID-Pred identified a set of 11 PCPs using a support vector machine and achieved 10-fold cross-validation and test accuracies of 99.53% and 97.80%, respectively. These findings could provide key insights into understanding the driving forces during the course of infection and assist in developing effective therapies.

Alterations in visceral adipose tissue (VAT) are closely linked to cardiometabolic abnormalities. The aim of this work is to define a metabolic signature in VAT of insulin resistance (IR) dependent on, and independent of, obesity. An untargeted UPLC-Q-Exactive metabolomic approach was carried out on the VAT of obese insulin-sensitive (IS) and insulin-resistant subjects (N = 11 and N = 25, respectively) and nonobese IS and IR subjects (N = 25 and N = 10, respectively). The VAT metabolome in obesity was defined among other things by changes in the metabolism of lipids, nucleotides, carbohydrates, and amino acids, whereas when combined with high IR, it affected the metabolism of 18 carbon fatty acyl-containing phospholipid species. A multimetabolite model created by glycerophosphatidylinositol (18:0); glycerophosphatidylethanolamine (18:2); glycerophosphatidylserine (18:0); and glycerophosphatidylcholine (18:0/18:1), (18:2/18:2), and (18:2/18:3) exhibited a highly predictive performance to identify the metabotype of "insulin-sensitive obesity" among obese individuals [area under the curve (AUC) 96.7% (91.9-100)] and within the entire study population [AUC 87.6% (79.0-96.2)]. We demonstrated that IR has a unique and shared metabolic signature dependent on, and independent of, obesity. For it to be used in clinical practice, these findings need to be validated in a more accessible sample, such as blood.

Bone proteomic studies using animal proxies and skeletonized human remains have delivered encouraging results in the search for potential biomarkers for precise and accurate post-mortem interval (PMI) and the age-at-death (AAD) estimation in medico-legal investigations. The development of forensic proteomics for PMI and AAD estimation is in critical need of research on human remains throughout decomposition, as currently the effects of both inter-individual biological differences and taphonomic alteration on the survival of human bone protein profiles are unclear. This study investigated the human bone proteome in four human body donors studied throughout decomposition outdoors. The effects of ageing phenomena (in vivo and post-mortem) and intrinsic and extrinsic variables on the variety and abundancy of the bone proteome were assessed. Results indicate that taphonomic and biological variables play a significant role in the survival of proteins in bone. Our findings suggest that inter-individual and inter-skeletal differences in bone mineral density (BMD) are important variables affecting the survival of proteins. Specific proteins survive better within the mineral matrix due to their mineral-binding properties. The mineral matrix likely also protects these proteins by restricting the movement of decomposer microbes. New potential biomarkers for PMI estimation and AAD estimation were identified. Future development of forensic bone proteomics should include standard measurement of BMD and target a combination of different biomarkers.

Intrinsic cardiorespiratory fitness (iCRF) indicates the CRF level in the sedentary state. However, even among sedentary individuals, a wide interindividual variability is observed in the iCRF levels, whose associated molecular characteristics are little understood. This study aimed to investigate whether serum and skeletal muscle metabolomics profiles are associated with iCRF, measured by maximal power output (MPO). Seventy sedentary young adults were submitted to venous blood sampling, a biopsy of the vastus lateralis muscle and iCRF assessment. Blood serum and muscle tissue samples were analyzed by proton nuclear magnetic resonance (1H NMR) spectroscopy. Metabolites related to iCRF were those supported by three levels of evidence: (1) correlation with iCRF, (2) significant difference between individuals with low and high iCRF, and (3) metabolite contribution to significant pathways associated with iCRF. From 43 serum and 70 skeletal muscle analyzed metabolites, iCRF was positively associated with levels of betaine, threonine, proline, ornithine, and glutamine in serum and lactate, fumarate, NADP+, and formate in skeletal muscle. Serum betaine and ornithine and skeletal muscle lactate metabolites explained 31.2 and 16.8%, respectively, of the iCRF variability in addition to body mass. The results suggest that iCRF in young adults is positively associated with serum and skeletal muscle metabolic levels, indicative of the amino acid and carbohydrate metabolism.

We performed quantitative metabolic phenotyping of blood plasma in parallel with cytokine/chemokine analysis from participants who were either SARS-CoV-2 (+) (n = 10) or SARS-CoV-2 (-) (n = 49). SARS-CoV-2 positivity was associated with a unique metabolic phenotype and demonstrated a complex systemic response to infection, including severe perturbations in amino acid and kynurenine metabolic pathways. Nine metabolites were elevated in plasma and strongly associated with infection (quinolinic acid, glutamic acid, nicotinic acid, aspartic acid, neopterin, kynurenine, phenylalanine, 3-hydroxykynurenine, and taurine; p < 0.05), while four metabolites were lower in infection (tryptophan, histidine, indole-3-acetic acid, and citrulline; p < 0.05). This signature supports a systemic metabolic phenoconversion following infection, indicating possible neurotoxicity and neurological disruption (elevations of 3-hydroxykynurenine and quinolinic acid) and liver dysfunction (reduction in Fischer's ratio and elevation of taurine). Finally, we report correlations between the key metabolite changes observed in the disease with concentrations of proinflammatory cytokines and chemokines showing strong immunometabolic disorder in response to SARS-CoV-2 infection.

Although cryopreservation is widely used in animal breeding, the technique is still suboptimal. The population of spermatozoa surviving the procedure experiences changes attributed to alteration in their redox regulation. In order to expand our knowledge regarding this particular aspect, the proteome in fresh and frozen thawed aliquots of equine spermatozoa was studied to identify the proteins most severely affected by the procedure. If alteration of redox regulation is a major factor explaining cryodamage, proteins participating in redox regulation should be principally affected. Using a split sample design, 30 ejaculates from 10 different stallions were analyzed as fresh spermatozoa, and another aliquot from the same ejaculate was analyzed as a frozen thawed sample. The proteome was studied under both conditions using UHPLC-MS/MS and bioinformatic analysis conducted to identify discriminant variables between both conditions. Data are available through the ProteomeXchange Consortium with identifier PXD022236. The proteins most significantly reduced were Aldo-keto reductase family 1 member B (p = 2.2 × 10-17) and Superoxide dismutase (Cu-Zn) (p = 4.7 × 10-14). This is the first time that SOD1 has been identified as a discriminating variable using bioinformatic analysis, where it was one of the most highly significantly different proteins seen between fresh and frozen thawed semen. This finding strongly supports the theory that alteration in redox regulation and oxidative stress is a major factor involved in cryodamage and suggests that control of redox regulation should be a major target to improve current cryopreservation procedures.