Diagnostic utility of N-terminal TMPP labels for unambiguous identification of clipped sites in therapeutic proteins.

Protein therapeutics are susceptible to clipping via enzymatic and nonenzymatic mechanisms that create neo-N-termini. Typically, neo-N-termini are identified by chemical derivatization of the N-terminal amine with (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) followed by proteolysis and mass spectrometric analysis. Detection of the TMPP-labeled peptide is achieved by mapping the peptide sequence to the product ion spectrum derived from collisional activation. The site-specific localization of the TMPP tag enables unambiguous determination of the true N-terminus or neo-N-termini. In addition to backbone product ions, TMPP reporter ions at m/z 573, formed via collision-induced dissociation, can be diagnostic for the presence of a processed N-termini. However, reporter ions generated by collision-induced dissociation may be uninformative because of their low abundance. We demonstrate a novel high-throughput LC-MS method for the facile generation of the TMPP reporter ion at m/z 533 and, in some instances m/z 590, upon electron transfer dissociation. We further demonstrate the diagnostic utility of TMPP labeled peptides derived from a total cell lysate shows high degree of specificity towards selective N-terminal labeling over labeling of lysine and tyrosine and highly-diagnostic Receiver Operating Characteristic's (ROC) of TMPP reporter ions of m/z 533 and m/z 590. The abundant generation of these reporters enables subsequent MS/MS by intensity and m/z-dependent triggering of complementary ion activation modes such as collision-induced dissociation, high-energy collision dissociation, or ultraviolet photo dissociation for subsequent peptide sequencing.

Urinary Metabolomics for the Prediction of Radiation-Induced Cardiac Dysfunction.

Survivors of acute radiation exposure are likely to experience delayed effects that manifest as injury in late-responding organs such as the heart. Non-invasive indicators of radiation-induced cardiac dysfunction are important in the prediction and diagnosis of this disease. In this study, we aimed to identify urinary metabolites indicative of radiation-induced cardiac damage by analyzing previously collected urine samples from a published study. The samples were collected from male and female wild-type (C57BL/6N) and transgenic mice constitutively expressing activated protein C (APCHi), a circulating protein with potential cardiac protective properties, who were exposed to 9.5 Gy of γ-rays. We utilized LC-MS-based metabolomics and lipidomics for the analysis of urine samples collected at 24 h, 1 week, 1 month, 3 months, and 6 months post-irradiation. Radiation caused perturbations in the TCA cycle, glycosphingolipid metabolism, fatty acid oxidation, purine catabolism, and amino acid metabolites, which were more prominent in the wild-type (WT) mice compared to the APCHi mice, suggesting a differential response between the two genotypes. After combining the genotypes and sexes, we identified a multi-analyte urinary panel at early post-irradiation time points that predicted heart dysfunction using a logistic regression model with a discovery validation study design. These studies demonstrate the utility of a molecular phenotyping approach to develop a urinary biomarker panel predictive of the delayed effects of ionizing radia-tion. It is important to note that no live mice were used or assessed in this study; instead, we focused solely on analyzing previously collected urine samples.

The Microbiome and Metabolomic Profile of the Transplanted Intestine with Long-Term Function.

We analyzed the fecal microbiome by deep sequencing of the 16S ribosomal genes and the metabolomic profiles of 43 intestinal transplant recipients to identify biomarkers of graft function. Stool samples were collected from 23 patients with stable graft function five years or longer after transplant, 15 stable recipients one-year post-transplant and four recipients with refractory rejection and graft loss within one-year post-transplant. Lactobacillus and Streptococcus species were predominant in patients with stable graft function both in the short and long term, with a microbiome profile consistent with the general population. Conversely, Enterococcus species were predominant in patients with refractory rejection as compared to the general population, indicating profound dysbiosis in the context of graft dysfunction. Metabolomic analysis demonstrated significant differences between the three groups, with several metabolites in rejecting recipients clustering as a distinct set. Our study suggests that the bacterial microbiome profile of stable intestinal transplants is similar to the general population, supporting further application of this non-invasive approach to identify biomarkers of intestinal graft function.

Renal Metabolome in Obese Mice Treated with Empagliflozin Suggests a Reduction in Cellular Respiration.

Sodium glucose cotransporter, type 2 inhibitors, such as Empagliflozin, are protective of the kidneys by unclear mechanisms. Our aim was to determine how Empagliflozin affected kidney cortical metabolome and lipidome in mice. Adult male TALLYHO mice (prone to obesity) were treated with a high-milk-fat diet, or this diet containing Empagliflozin (0.01%), for 8 weeks. Targeted and untargeted metabolomics and lipidomics were conducted on kidney cortex by liquid chromatography followed by tandem mass-spectroscopy. Metabolites were statistically analyzed by MetaboAnalyst 5.0, LipidSig (lipid species only) and/or CEU Mass Mediator (untargeted annotation). In general, volcano plotting revealed oppositely skewed patterns for targeted metabolites (primarily hydrophilic) and lipids (hydrophobic) in that polar metabolites showed a larger number of decreased species, while non-polar (lipids) had a greater number of increased species (>20% changed and/or raw p-value < 0.05). The top three pathways regulated by Empagliflozin were urea cycle, spermine/spermidine biosynthesis, and aspartate metabolism, with an amino acid network being highly affected, with 14 of 20 classic amino acids down-regulated. Out of 75 changed polar metabolites, only three were up-regulated, i.e., flavin mononucleotide (FMN), uridine, and ureidosuccinic acid. Both FMN and uridine have been shown to be protective of the kidney. Scrutiny of metabolites of glycolysis/gluconeogenesis/Krebs cycle revealed a 20−45% reduction in several species, including phosphoenolpyruvate (PEP), succinate, and malic acid. In contrast, although overall lipid quantity was not higher, several lipid species were increased by EMPA, including those of the classes, phosphatidic acids, phosphatidylcholines, and carnitines. Overall, these analyses suggest a protection from extensive metabolic load and the corresponding oxidative stress with EMPA in kidney. This may be in response to reduced energy demands of the proximal tubule as a result of inhibition of transport and/or differences in metabolic pools available for metabolism.

Pharmacokinetic and metabolomic studies with a BIO 300 Oral Powder formulation in nonhuman primates.

BIO 300, a pharmaceutical formulation of genistein, is being developed as a radiation countermeasure to treat hematopoietic acute radiation syndrome (H-ARS) and the delayed effects of acute radiation exposure (DEARE). Several studies have affirmed its safety and efficacy in alleviating the damaging effects of ionizing radiation. However, dose optimization of any drug has always been an important area of research because unnecessarily high drug doses may result in serious complications. In this study, we assessed the pharmacokinetics (PK) and metabolic profiles of two different doses of a novel solid-dosage formulation of BIO 300 (BIO 300 Oral Powder; 100 mg/kg and 200 mg/kg), when administered orally to nonhuman primates (NHPs). While the Tmax values of both doses remained the same, the area under the curve at 48 h (AUC0-48) was tripled by doubling the dose. Additionally, we monitored serum samples for global metabolomic/lipidomic changes using high resolution mass spectrometry followed by functional pathway analysis prior to and at various time points up to 48 h post drug administration. Interestingly, the metabolomic profiles of sera from NHPs that received the lower dose demonstrated a transient perturbation in numerous metabolites between the 4 and 12 h time points. Eventually, the metabolite abundance reverted to near-normal by 48 h. These study results are consistent with our previous studies focused on the PK and metabolomic analysis for parenteral and oral aqueous nanosuspension formulations of BIO 300. This study affirms that administration of a single dose of up to 200 mg/kg of BIO 300 Oral Powder is safe in NHPs and conferred no metabolomic-mediated safety features.

An optimized method for the isolation of urinary extracellular vesicles for molecular phenotyping: detection of biomarkers for radiation exposure.

BACKGROUND: Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications across clinical research, including monitoring radiation exposure. A key limitation to their implementation is minimal standardization in EV isolation and analytical methods. Further, most urinary EV isolation protocols necessitate large volumes of sample. This study aimed to compare and optimize isolation and analytical methods for EVs from small volumes of urine. METHODS: 3 EV isolation methods were compared: ultracentrifugation, magnetic bead-based, and size-exclusion chromatography from 0.5 mL or 1 mL of rat and human urine. EV yield and mass spectrometry signals (Q-ToF and Triple Quad) were evaluated from each method. Metabolomic profiling was performed on EVs isolated from the urine of rats exposed to ionizing radiation 1-, 14-, 30- or 90-days post-exposure, and human urine from patients receiving thoracic radiotherapy for the treatment of lung cancer pre- and post-treatment. RESULTS: Size-exclusion chromatography is the preferred method for EV isolation from 0.5 mL of urine. Mass spectrometry-based metabolomic analyses of EV cargo identified biochemical changes induced by radiation, including altered nucleotide, folate, and lipid metabolism. We have provided standard operating procedures for implementation of these methods in other laboratories. CONCLUSIONS: We demonstrate that EVs can be isolated from small volumes of urine and analytically investigated for their biochemical contents to detect radiation induced metabolomic changes. These findings lay a groundwork for future development of methods to monitor response to radiotherapy and can be extended to an array of molecular phenotyping studies aimed at characterizing EV cargo.

Small Molecule Signatures of Mice Lacking T-cell p38 Alternate Activation, a Model for Immunosuppression Conditions, after Total-Body Irradiation.

Several diagnostic biodosimetry tools have been in development that may aid in radiological/nuclear emergency responses. Of these, correlating changes in non-invasive biofluid small-molecule signatures to tissue damage from ionizing radiation exposure show promise for inclusion in predictive biodosimetry models. Integral to dose reconstruction has been determining how genotypic variation in the general population will affect model performance. Here, we used a mouse model that lacks the T-cell receptor specific alternative p38 pathway [p38αßY323F, double knock-in (DKI) mice] to determine how attenuated autoimmune and inflammatory responses may affect dose reconstruction. We exposed adult male DKI mice (8-10 weeks old) to 2 and 7 Gy in parallel with wild-type mice and assessed perturbations in urine (days 1, 3, 7) and serum (day 1) using a global metabolomics approach. A multidimensional scaling plot showed excellent separation of radiation-exposed groups in wild-type mice with slightly dampened responses in DKI mice. Validated metabolite panels were developed for urine [N6,N6,N6-trimethyllysine (TML), N1-acetylspermidine, spermidine, carnitine, acylcarnitine C21H35NO5, aminohippuric acid] and serum [phenylalanine, glutamine, propionylcarnitine, lysophosphatidylcholine (LysoPC 14:0), LysoPC (22:5)] to determine the area under the receiver operating characteristic curve (AUROC). For both urine and serum, excellent sensitivity and specificity (AUROC > 0.90) was observed for 0 Gy vs. 7 Gy groups irrespective of genotype using identical metabolite panels. Similarly, excellent to fair classification (AUROC > 0.75) was observed for ≤2 Gy vs. 7 Gy mice for both genotypes, however, model performance declined (AUROC < 0.75) between genotypes after irradiation. Overall, these results suggest immunosuppression should not compromise small molecule multiplex panels used in dose reconstruction for biodosimetry.

Short-term metabolic disruptions in urine of mouse models following exposure to low doses of oxygen ion radiation.

Molecular alterations as a result of exposure to low doses of high linear energy transfer (LET) radiation can have deleterious short- and long-term consequences on crew members embarking on long distance space missions. Oxygen ions (16O) are among the high LET charged particles that make up the radiation environment inside a vehicle in deep space. We used mass spectrometry-based metabolomics to characterize urinary metabolic profiles of male C57BL/6J mice exposed to a single dose of 0.1, 0.25 and 1.0 Gy of 16O (600 MeV/n) at 10 and 30 days post-exposure to delineate radiation-induced metabolic alterations. We recognized a significant down regulation of several classes of metabolites including cresols and tryptophan metabolites, ketoacids and their derivatives upon exposure to 0.1 and 0.25 Gy after 10 days. While some of these changes reverted to near normal by 30 days, some metabolites including p-Cresol sulfate, oxalosuccinic acid, and indoxylsulfate remained dysregulated at 30 days, suggesting long term prognosis on metabolism. Pathway analysis revealed a long-term dysregulation in multiple pathways including tryptophan and porphyrin metabolism. These results suggest that low doses of high-LET charged particle irradiation may have long-term implications on metabolic imbalance.

Longitudinal metabolic alterations in plasma of rats exposed to low doses of high linear energy transfer radiation.

Astronauts embarking on deep space missions are at high risk of long-term exposure to low doses of high linear energy transfer (LET) radiation, which can contribute to the development of cancer and multiple degenerative diseases. However, long term effects of exposure to low doses of high LET radiation in plasma metabolite profiles have not been elucidated. We utilized an untargeted metabolomics and lipidomics approach to analyze plasma obtained from adult male Long Evans rats to determine the longitudinal effects of low-dose proton and low-dose oxygen ion whole-body irradiation on metabolic pathways. Our findings reveal that radiation exposure induced modest changes in the metabolic profiles in plasma, 7 months after exposure. Furthermore, we identified some common metabolite dysregulations between protons and oxygen ions, which may indicate a similar mechanism of action for both radiation types.

Plasma metabolite biomarkers predictive of radiation induced cardiotoxicity.

PURPOSE: Although advancements in cancer treatments using radiation therapy (RT) have led to improved outcomes, radiation-induced heart disease (RIHD) remains a significant source of morbidity and mortality in survivors of cancers in the chest. Currently, there are no diagnostic tests in clinical use due to a lack of understanding of the natural history and mechanisms of RIHD development. Few studies have examined the utility of using metabolomics to prospectively identify cancer survivors who are at risk of developing cardiotoxicity. METHODS: We analyzed plasma and left ventricle heart tissue samples collected from a cohort of male Sprague Dawley (SD) rats that were either sham irradiated or received fractionated doses (9 Gy per day × 5 days) of targeted X-ray radiation to the heart. Metabolomic and lipidomic analyses were utilized as a correlative approach for delineation of novel biomarkers associated with radiation-induced cardiac toxicity. Additionally, we used high-resolution mass spectrometry to examine the metabolomic profiles of plasma samples obtained from patients receiving high dose thoracic RT for esophageal cancer. RESULTS: Metabolic alterations in the rat model and patient plasma profiles, showed commonalities of radiation response that included steroid hormone biosynthesis and vitamin E metabolism. Alterations in patient plasma profiles were used to develop classification algorithms predictive of patients at risk of developing RIHD. CONCLUSION: Herein, we report the feasibility of developing a metabolomics-based biomarker panel that is associated with adverse outcomes of cardiac function in patients who received RT for the treatment of esophageal cancer.

Alterations in Tissue Metabolite Profiles with Amifostine-Prophylaxed Mice Exposed to Gamma Radiation.

Acute exposure to high-dose ionizing irradiation has the potential to severely injure the hematopoietic system and its capacity to produce vital blood cells that innately serve to ward off infections and excessive bleeding. Developing a medical radiation countermeasure that can protect individuals from the damaging effects of irradiation remains a significant, unmet need and an area of great public health interest and concern. Despite significant advancements in the field of radiation countermeasure development to find a nontoxic and effective prophylactic agent for acute radiation syndrome, no such drug has yet been approved by the Food and Drug Administration. This study focuses on examining the metabolic corrections elicited by amifostine, a potent radioprotector, on tissues of vital body organs, such as the heart, spleen, and kidney. Our findings indicate that prophylaxis with this drug offers significant protection against potentially lethal radiation injury, in part, by correction of radiation-induced metabolic pathway perturbations.

Metabolomic studies in tissues of mice treated with amifostine and exposed to gamma-radiation.

Although multiple radioprotectors are currently being investigated preclinically for efficacy and safety, few studies have investigated concomitant metabolic changes. This study examines the effects of amifostine on the metabolic profiles in tissues of mice exposed to cobalt-60 total-body gamma-radiation. Global metabolomic and lipidomic changes were analyzed using ultra-performance liquid chromatography (UPLC) quadrupole time-of-flight mass spectrometry (QTOF-MS) in bone marrow, jejunum, and lung samples of amifostine-treated and saline-treated control mice. Results demonstrate that radiation exposure leads to tissue specific metabolic responses that were corrected in part by treatment with amifostine in a drug-dose dependent manner. Bone marrow exhibited robust responses to radiation and was also highly responsive to protective effects of amifostine, while jejunum and lung showed only modest changes. Treatment with amifostine at 200 mg/kg prior to irradiation seemed to impart maximum survival benefit, while the lower dose of 50 mg/kg offered only limited survival benefit. These findings show that the administration of amifostine causes metabolic shifts that would provide an overall benefit to radiation injury and underscore the utility of metabolomics and lipidomics to determine the underlying physiological mechanisms involved in the radioprotective efficacy of amifostine. This approach may be helpful in identifying biomarkers for radioprotective efficacy of amifostine and other countermeasures under development.

Salivary Metabolomics of Total Body Irradiated Nonhuman Primates Reveals Long-Term Normal Tissue Responses to Radiation.

PURPOSE: To identify metabolomic biomarkers of acute radiation exposure in saliva that show time-dependent changes. METHODS AND MATERIALS: Nonhuman primates were exposed to 4 Gy of total body irradiation with γ-rays. Saliva was collected from 7 animals twice before and at days 1, 3, 5, 7, 15, 21, 28, and 60 after irradiation. Profiling was conducted with liquid chromatography time-of-flight mass spectrometry. Multivariate data analysis and potential biomarker identification was conducted through random Forests and the software MetaboAnalyst. Candidate biomarkers were validated through tandem mass spectrometry, and receiver operating characteristic curves were constructed to show the diagnostic ability of the signature over time. RESULTS: Untargeted metabolomic analysis revealed significant and persistent effects up to the 60 days evaluated in this study. Biomarkers spanning primarily amino acids and nucleotides were identified, with a significant number showing long-term responses. Fifteen biomarkers showed high statistical significance in the first week after irradiation and 16 at >7 days after irradiation (false discovery rate-adjusted P < .05). The combination of the biomarkers in a single biosignature was able to accurately show the diagnostic ability of the signature in a binary classifier system with receiver operating characteristic curves. CONCLUSIONS: Radiation can alter the metabolome in saliva, and metabolomics could effectively be used to monitor radiation responses, as a biodosimetry method, in the event of a radiological incident. Saliva metabolomics also has potential relevance in a clinical setting.

Metabolomic Analysis of Liver Tissues for Characterization of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) causes more than half a million annual deaths worldwide. Understanding the mechanisms contributing to HCC development is highly desirable for improved surveillance, diagnosis, and treatment. Liver tissue metabolomics has the potential to reflect the physiological changes behind HCC development. Also, it allows identification of biomarker candidates for future evaluation in biofluids and investigation of racial disparities in HCC. Tumor and nontumor tissues from 40 patients were analyzed by both gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) platforms to increase the metabolome coverage. The levels of the metabolites extracted from solid liver tissue of the HCC area and adjacent non-HCC area were compared. Among the analytes detected by GC-MS and LC-MS with significant alterations, 18 were selected based on biological relevance and confirmed metabolite identification. These metabolites belong to TCA cycle, glycolysis, purines, and lipid metabolism and have been previously reported in liver metabolomic studies where high correlation with HCC progression is implied. We demonstrated that metabolites related to HCC pathogenesis can be identified through liver tissue metabolomic analysis. Additionally, this study has enabled us to identify race-specific metabolites associated with HCC.