Development and Validation of a Bioanalytical UHPLC-MS/MS Method Applied to Murine Liver Tissue for the Determination of Indocyanine Green Loaded in H-Ferritin Nanoparticles

Indocyanine green (ICG) is one of the most commonly used fluorophores in near-infrared fluorescence-guided techniques. However, the molecule is prone to form aggregates in saline solution with a limited photostability and a moderate fluorescence yield. ICG was thus formulated using protein-based nanoparticles of H-ferritin (HFn) in order to generate a new nanostructure, HFn-ICG. In this study, an ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) system was employed to develop and validate the quantitative analysis of ICG in liver tissue samples from HFn-ICG-treated mice. To precipitate HFn, cold acetone in acidic solution at pH 5.0 was used. The processed liver samples were injected into the UHPLC-MS/MS system for analysis using the positive electrospray ionization mode. Chromatographic separation was achieved on a Waters Acquity UPLC® HSS T3 Column (1.8 μm, 2.1 × 100 mm) with 0.1% formic acid and acetonitrile as the mobile phase with gradient elution. The selected reaction monitoring transitions of m/z 753 →m/z 330 and m/z 827 →m/z 330 were applied for ICG and IR-820 (the internal standard, IS), respectively. The method was selective and linear over a concentration range of 50–1,500 ng/ml. The method was validated for sensitivity, accuracy, precision, extraction recovery, matrix effect, and stability in liver tissue homogenates. ICG extraction recoveries ranged between 85 and 108%. The intra- and inter-day precisions were less than 6.28%. The method was applied to a bio-distribution study to compare the amount of ICG levels from mice treated with HFn-ICG and free ICG. The analyses of the homogenate samples from the two types of treatment showed that the concentration levels of ICG is approximately six-fold higher than those of free ICG (1,411 ± 7.62 ng/ml vs. 235 ± 26.0 ng/ml) at 2 h post injection.

Monitoring one-carbon metabolism by mass spectrometry to assess liver function and disease

Precision medicine promises to overcome the constraints of the traditional “one-for-all” healthcare approach through a clear understanding of the molecular features of a disease, allowing for innovative and tailored treatments. State-of-the-art proteomics has the potential to accurately explore the human proteome to identify, quantify, and characterize proteins associated with disease progression. There is a pressing need for informative biomarkers to diagnose liver disease early in its course to prevent severe disease for which no efficient treatment is yet available. Here, we propose the concept of a cellular pathway as a functional biomarker, whose monitorization may inform normal and pathological status. We have developed a standardized targeted selected-reaction monitoring assay to detect and quantify 13 enzymes of one-carbon metabolism (1CM). The assay is compliant with Clinical Proteomics Tumor Analysis Consortium (CPTAC) guidelines and has been included in the protein quantification assays that can be accessed through the assay portal at the CPTAC web page. To test the feasibility of the assay, we conducted a retrospective, proof-of-concept study on a collection of liver samples from healthy controls and from patients with cirrhosis or hepatocellular carcinoma (HCC). Our results indicate a significant reconfiguration of 1CM upon HCC development resulting from a process that can already be identified in cirrhosis. Our findings indicate that the systematic and integrated quantification of 1CM enzymes is a promising cell function-based biomarker for patient stratification, although further experiments with larger cohorts are needed to confirm these findings.

Time-Dependent Analysis of Plasmalogens in the Hippocampus of an Alzheimer’s Disease Mouse Model: A Role of Ethanolamine Plasmalogen

Plasmalogens are alkenyl-acyl glycerophospholipids and decreased in post-mortem Alzheimer’s disease (AD) brains. The aim of this study is to investigate the time-dependent changes of plasmalogens in the hippocampus of an AD model mouse (J20). Plasmalogen levels at 3, 6, 9, 12 and 15 months were analyzed by liquid-chromatography-targeted-multiplexed-selected-reaction-monitoring-tandem-mass-spectrometry (LC-SRM/MS). Reactive oxygen species (ROS) levels were evaluated using dichlorofluorescein diacetate (DCF-DA). Plasmalogen synthesizing enzyme glycerone-phosphate O-acyltransferase (GNPAT) and late endosome marker Rab7 levels were quantified by Western blotting. GNPAT localization, changes of neuronal and glial cell numbers were evaluated by immunostaining. Compared to wild-type mice (WT), total plasmalogen-ethanolamine, but not plasmalogen-choline levels, were increased at 9 months and subsequently decreased at 15 months in J20 mice. A principal component analysis of plasmalogen-ethanolamine species could separate WT and J20 mice both at 9 and 15 months. Both GNPAT and Rab7 protein were increased in J20 mice at 9 months, whereas GNPAT was decreased at 15 months. ROS levels were increased in J20 mice except for 9 months. Our results suggest that increased plasmalogen-ethanolamine could counteract ROS levels and contribute to the phagocytosis process in J20 mice at 9 months. Such results might indicate a transient protective response of plasmalogen-ethanolamine in AD conditions.

Identification of cerebrospinal fluid biomarkers for parkinsonism using a proteomics approach

The aim of our study was to investigate cerebrospinal fluid (CSF) tryptic peptide profiles as potential diagnostic biomarkers for the discrimination of parkinsonian disorders. CSF samples were collected from individuals with parkinsonism, who had an uncertain diagnosis at the time of inclusion and who were followed for up to 12 years in a longitudinal study. We performed shotgun proteomics to identify tryptic peptides in CSF of Parkinson’s disease (PD, n = 10), multiple system atrophy patients (MSA, n = 5) and non-neurological controls (n = 10). We validated tryptic peptides with differential levels between PD and MSA using a newly developed selected reaction monitoring (SRM) assay in CSF of PD (n = 46), atypical parkinsonism patients (AP; MSA, n = 17; Progressive supranuclear palsy; n = 8) and non-neurological controls (n = 39). We identified 191 tryptic peptides that differed significantly between PD and MSA, of which 34 met our criteria for SRM development. For 14/34 peptides we confirmed differences between PD and AP. These tryptic peptides discriminated PD from AP with moderate-to-high accuracy. Random forest modelling including tryptic peptides plus either clinical assessments or other CSF parameters (neurofilament light chain, phosphorylated tau protein) and age improved the discrimination of PD vs. AP. Our results show that the discovery of tryptic peptides by untargeted and subsequent validation by targeted proteomics is a suitable strategy to identify potential CSF biomarkers for PD versus AP. Furthermore, the tryptic peptides, and corresponding proteins, that we identified as differential biomarkers may increase our current knowledge about the disease-specific pathophysiological mechanisms of parkinsonism.

Number of detected proteins as the function of the sensitivity of proteomic technology in human liver cells

The main goal of the Russian part of C-HPP is to detect and functionally annotate missing proteins (PE2-PE4) encoded by human chromosome 18. However, identifying such proteins in a complex biological mixture using mass spectrometry (MS)-based methods is difficult due to the insufficient sensitivity of proteomic analysis methods. In this study, we determined the proteomic technology sensitivity using a standard set of UPS1 proteins as an example. The results revealed that 100% of proteins in a mixture could only be identified at a concentration of at least 10-9 M. The decrease in concentration leads to protein losses associated with technology sensitivity, and no UPS1 protein is detected at a concentration of 10-13 M. Therefore, two-dimensional fractionation of samples was applied to improve sensitivity. The human liver tissue was examined by selected reaction monitoring and shotgun methods of MS analysis using one-dimensional and two-dimensional fractionation to identify the proteins encoded by human chromosome 18. A total of 134 proteins were identified. The overlap between proteomic and transcriptomic data in human liver tissue was ~50%. This weak convergence is due to the low sensitivity of proteomic technology compared to transcriptomic approaches. Data is available via ProteomeXchange with identifier PXD026997.

Targeted Selected Reaction Monitoring Verifies Histology Specific Peptide Signatures in Epithelial Ovarian Cancer

Epithelial ovarian cancer (OC) is a disease with high mortality due to vague early clinical symptoms. Benign ovarian cysts are common and accurate diagnosis remains a challenge because of the molecular heterogeneity of OC. We set out to investigate whether the disease diversity seen in ovarian cyst fluids and tumor tissue could be detected in plasma. Using existing mass spectrometry (MS)-based proteomics data, we constructed a selected reaction monitoring (SRM) assay targeting peptides from 177 cancer-related and classical proteins associated with OC. Plasma from benign, borderline, and malignant ovarian tumors were used to verify expression (n = 74). Unsupervised and supervised multivariate analyses were used for comparisons. The peptide signatures revealed by the supervised multivariate analysis contained 55 to 77 peptides each. The predictive (Q2) values were higher for benign vs. low-grade serous Q2 = 0.615, mucinous Q2 = 0.611, endometrioid Q2 = 0.428 and high-grade serous Q2 = 0.375 (stage I–II Q2 = 0.515; stage III Q2 = 0.43) OC compared to benign vs. all malignant Q2 = 0.226. With targeted SRM MS we constructed a multiplexed assay for simultaneous detection and relative quantification of 185 peptides from 177 proteins in only 20 µL of plasma. With the approach of histology-specific peptide patterns, derived from pre-selected proteins, we may be able to detect not only high-grade serous OC but also the less common OC subtypes.

Immunoprecipitation-targeted proteomics assays facilitate rational development of SARS-CoV-2 serological diagnostics

Current design of serological tests employs conservative immunoassay approaches and is often focused on convenience, speed of manufacturing, and affordability. Limitations of such serological tests include semi-quantitative measurements, lack of standardization, potential cross-reactivity, and inability to distinguish between antibody subclasses. As a result of cross-reactivity, diagnostic specificity of serological antibody tests may not be sufficiently high to enable screening of the general asymptomatic populations for the acquired immunity against low-prevalence infectious diseases, such as COVID-19. Likewise, lack of a single standard for assay calibration limits inter-laboratory and international standardization of serological tests. In this study, we hypothesize that combination of immunoaffinity enrichments with targeted mass spectrometry measurements would enable rational design of serology diagnostics of infectious diseases, such as COVID-19. The same instrumental platform allows for sensitive and specific measurements of viral protein antigens, as wells as anti-viral antibodies circulating in human serum. Our proof-of-concept immunoprecipitation - parallel reaction monitoring (IP-PRM) assays quantified NCAP_SARS2 protein with a limit of detection of 313 pg/mL in serum. In addition, a multiplex IP-selected reaction monitoring (IP-SRM) assay facilitated differential quantification of anti-SARS-CoV-2 antibody isotypes and subclasses in patient sera. Simultaneous evaluation of numerous antigen-antibody subclass combinations revealed a receptor-binding domain (RBD)-IgG1 as a combination with the highest diagnostic specificity and sensitivity. Anti-RBD IgG1, IgG3, IgM and IgA1 subclasses, but not IgG2, IgG4 and IgA2, were found elevated in COVID-19-positive sera. Synthetic heavy isotope-labeled peptide internal standards as calibrators revealed elevated anti-RBD IgG1 in positive (510-6700 ng/mL; 0.02-0.22% of total serum IgG1) versus negative sera (60 [interquartile range 41-81] ng/mL). Likewise, anti-RBD IgM was elevated in positive (190-510 ng/mL; 0.06-0.16% of total serum IgM) versus negative sera (76 [31-108] ng/mL). Further validation of immunoprecipitation-targeted proteomics assays as a platform for serological assays will facilitate standardization and improvement of the existing serological tests, enable rational design of novel tests, and offer tools for comprehensive investigation of antibody isotype and subclass cooperation in immunity response.

Pharmacokinetic study of hypaphorine, a potential agent for treating osteoclast-based bone loss, on rats using LC-MS/MS

Aim and Objective: A high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of hypaphorine, a potential agent for treating osteoclast-based bone loss, was developed and valadated in rat plasma. Materials and Methods: Plasma samples were pretreated by the protein precipitation. Chromatographic separation was performed using an Inertsil ODS-3 column (50 mm × 4.6 mm, 5 μm). The mobile phase consisted of water (containing 0.1% formic acid) and acetonitrile in a gradient mode at a flow rate of 0.5 mL/min. The acquisition was carried out in selected reaction monitoring (SRM) of the transitions from protonated precursor ion [M + H]+ to the particular daughter ion and the mass transitions of hypaphorine and IS were 247 → 188 and m/z 219 → 188, respectively. The method was validated in terms of selectivity, linearity, accuracy and precision, extraction recovery and matrix effect, stability and carryover. Results: It showed good linearity over the range of 1-2000 ng/mL (R2 = 0.9978). The intra-batch accuracy was within 93.95-105.81% and the precision was within 4.92-11.53%. The inter-batch accuracy was within 96.18-100.39% with a precision of 6.22-11.23%. The extraction recovery and matrix factors were acceptable. Conclusion: The simple and rapid method was successfully applied to the pharmacokinetics study in rats following oral administration of hypaphorine at the doses of 0.5, 1.5, and 4.5 mg/kg.