Parallel Reaction Monitoring Mass Spectrometry Research Articles

Small RNA-regulated expression of efflux pump affects tigecycline resistance and heteroresistance in clinical isolates of Klebsiella pneumoniae

Tigecycline and the newly Food and Drug Administration-approved tetracyclines, including eravacycline and omadacycline, are regarded as last-resort treatments for multidrug-resistant Enterobacterales. However, tigecycline resistance in Klebsiella pneumoniae has increased, especially the underlying mechanism of heteroresistance is unclear. This study aimed to elucidate the mechanisms underlying tigecycline resistance and heteroresistance in clinical K. pneumoniae isolates. A total of 153 clinical K. pneumoniae isolates were collected, and identified 15 tigecycline-resistant and three tigecycline-heteroresistant isolates using broth microdilution and population analysis profile methods, respectively. Total RNAs from K. pneumoniae ATCC13883 and the laboratory-induced tigecycline-resistant strain were extracted and sequenced on an Illumina platform. Differentially expressed genes and regulatory small RNAs (sRNAs) were analyzed and validated in clinical isolates of K. pneumoniae using quantitative real-time PCR. RNA sequencing results showed that mdtABC efflux pump genes were significantly upregulated in the tigecycline-resistant strains. Overexpression of mdtABC was observed in a clinical K. pneumoniae isolate, which increased tigecycline minimum inhibitory concentrations (MICs) and was involved in tigecycline heteroresistance. Sequencing analysis of sRNA demonstrated that candidate sRNA-120 directly interacted with the mdtABC operon and was downregulated in tigecycline-resistant strains. We generated an sRNA-120 deletion mutation strain and a complemented strain of K. pneumoniae. The sRNA-120 deletion strain displayed increased mRNA levels of mdtA, mdtB, and mdtC and an increase in MICs of tigecycline. The complemented strain of sRNA-120 restored the mRNA levels of these genes and the susceptibility to tigecycline. RNA antisense purification and parallel reaction monitoring mass spectrometry were performed to verify the interactions between sRNA-120 and mdtABC. Collectively, our study highlights that the post-transcriptional repression of mdtABC through sRNA-120 may provide an additional layer of efflux pump gene expression control, which is important for resistance and heteroresistance in clinical K. pneumoniae isolates.

Abstract 936: Targeted proteomics and next-generation sequencing (NGS) for biomarker discovery in metastatic colorectal cancer (mCRC)

Abstract Background: Multi-omics analysis is widely used in cancer diagnosis, prognosis and prediction of treatment response. Here we introduce the use of targeted proteomics coupled with NGS as tools to assess oncogenic alterations along with quantitative protein expression in patients with mCRC. Our proteomics panel included proteins that are antibody drug conjugates (ADCs) targets (ERBB 1-4, TROP2, CEACAM5 etc.) as well as proteins involved in sensitivity to ADC payload (SLFN11, drug efflux pump, TOP1 etc.). Methods: The HORIZON III trial (NCT00384176) evaluated FOLFOX + bevacizumab or cediranib as first-line treatment in patients with mCRC (Schmoll J Clin Oncol 2012). In the current analysis, a subset of samples from the HORIZON III trial were assessed for molecular alterations using NGS and proteomics. Pre-existing HER2 IHC scores were used as previously referenced (Cecchi J Clin Oncol 2023). Samples were processed (n=135) with an automated extraction and library preparation method, then sequenced with both a Whole Exome and targeted-custom gene panel. NGS analysis had a success rate of 116/135 samples (87.3%). Absolute quantitation of protein targets was performed using parallel reaction monitoring (PRM) mass spectrometry after collecting tumor epithelial cells by laser microdissection and punch biopsies (n=60) followed by trypsin digestion. Results: The NGS analysis showed that patients had mutated RAS (50%), BRAF (10%), PIK3CA (16%), and TMB-H (18%) and MSI-H (5%) status. A distinguished pattern of mutual exclusivity among KRAS, NRAS, and BRAF mutations is evident. The HER2-positive patients were wildtype RAS, BRAF, and PIK3CA, MSS and TMB-L. Samples that were IHC HER2-positive matched those with NGS ERBB2 amplification. Using PRM, nearly all the evaluable samples had detectable HER2. HER2 IHC 3+ sample showed the highest protein expression at 30,000 amol/ug (equivalent to 1M receptors/cell). Within the HER2-negative patients, HER2 protein expression showed broad dynamic range. CEACAM5 and HER2 are the only ADC targets that reached the level of 1M receptors/cell. Highest TROP2 protein expression is 5 times lower than the highest HER2 value. Conclusions: Our preliminary findings for this cohort indicate the benefit of the use of multi-modal methodologies to inform better strategies for patient stratification and precision medicine, though further assessment of proteogenomic characteristics is needed. This data may inform future clinical development and guide patient eligibility to clinical trials and possible treatment sequencing for ADCs. Citation Format: William J. Kelly, Barrett Nuttall, Marlon Rebelatto, Marcus Schick, Armin Meier, Steve Sweet, Beom-Jun Kim, David Chain, Yeoun Jin Kim, Zhongwu Lai, Ying Wang, Todd H. Creasy, Ramya Gurrapu, Sakshi Gulati, Alice Barkell, Brian Dougherty, Danielle Carroll, Fabiola Cecchi. Targeted proteomics and next-generation sequencing (NGS) for biomarker discovery in metastatic colorectal cancer (mCRC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 936.

Multiplatform tear proteomic profiling reveals novel non-invasive biomarkers for diabetic retinopathy.

To investigate a comprehensive proteomic profile of the tear fluid in patients with diabetic retinopathy (DR) and further define non-invasive biomarkers. A cross-sectional, multicentre study that includes 46 patients with DR, 28 patients with diabetes mellitus (DM), and 30 healthy controls (HC). Tear samples were collected with Schirmer strips. As for the discovery set, data-independent acquisition mass spectrometry was used to characterize the tear proteomic profile. Differentially expressed proteins between groups were identified, with gene ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes enrichment analysis further developed. Classifying performance of biomarkers for distinguishing DR from DM was compared by the combination of three machine-learning algorithms. The selected biomarker panel was tested in the validation cohort using parallel reaction monitoring mass spectrometry. Among 3364 proteins quantified, 235 and 88 differentially expressed proteins were identified for DR when compared to HC and DM, respectively, which were fundamentally related to retina homeostasis, inflammation and immunity, oxidative stress, angiogenesis and coagulation, metabolism, and cellular adhesion processes. The biomarker panel consisting of NAD-dependent protein deacetylase sirtuin-2 (SIR2), amine oxidase [flavin-containing] B (AOFB), and U8 snoRNA-decapping enzyme (NUD16) exhibited the best diagnostic performance in discriminating DR from DM, with AUCs of 0.933 and 0.881 in the discovery and validation set, respectively. Tear protein dysregulation is comprehensively revealed to be associated with DR onset. The combination of tear SIR2, AOFB, and NUD16 can be a novel potential approach for non-invasive detection or pre-screening of DR. Chinese Clinical Trial Registry Identifier: ChiCTR2100054263. https://www.chictr.org.cn/showproj.html?proj=143177 . Date of registration: 2021/12/12.

Targeted Mass Spectrometry of Longitudinal Patient Sera Reveals LTBP1 as a Potential Surveillance Biomarker for High-Grade Serous Ovarian Carcinoma.

High-grade serous ovarian carcinoma (HGSC) is the most prevalent subtype of epithelial ovarian cancer. The combination of a high rate of recurrence and novel therapies in HGSC necessitates an accurate assessment of the disease. Currently, HGSC response to treatment and recurrence are monitored via immunoassay of serum levels of the glycoprotein CA125. CA125 levels predictably rise at HGSC recurrence; however, it is likely that the disease is progressing even before it is detectable through CA125. This may explain why treating solely based on CA125 increase has not been associated with improved outcomes. Thus, additional biomarkers that monitor HGSC progression and cancer recurrence are needed. For this purpose, we developed a scheduled parallel reaction monitoring mass spectrometry (PRM-MS) assay for the quantification of four previously identified HGSC-derived glycopeptides (from proteins FGL2, LGALS3BP, LTBP1, and TIMP1). We applied the assay to quantify their longitudinal expression profiles in 212 serum samples taken from 34 HGSC patients during disease progression. Analyses revealed that LTBP1 best-mirrored tumor load, dropping as a result of cancer treatment in 31 out of 34 patients and rising at HGSC recurrence in 28 patients. Additionally, LTBP1 rose earlier during remission than CA125 in 11 out of 25 platinum-sensitive patients with an average lead time of 116.4 days, making LTBP1 a promising candidate for monitoring of HGSC recurrence.

Quantification of putative ovarian cancer serum protein biomarkers using a multiplexed targeted mass spectrometry assay

BackgroundOvarian cancer is the most lethal gynecologic malignancy in women, and high-grade serous ovarian cancer (HGSOC) is the most common subtype. Currently, no clinical test has been approved by the FDA to screen the general population for ovarian cancer. This underscores the critical need for the development of a robust methodology combined with novel technology to detect diagnostic biomarkers for HGSOC in the sera of women. Targeted mass spectrometry (MS) can be used to identify and quantify specific peptides/proteins in complex biological samples with high accuracy, sensitivity, and reproducibility. In this study, we sought to develop and conduct analytical validation of a multiplexed Tier 2 targeted MS parallel reaction monitoring (PRM) assay for the relative quantification of 23 putative ovarian cancer protein biomarkers in sera.MethodsTo develop a PRM method for our target peptides in sera, we followed nationally recognized consensus guidelines for validating fit-for-purpose Tier 2 targeted MS assays. The endogenous target peptide concentrations were calculated using the calibration curves in serum for each target peptide. Receiver operating characteristic (ROC) curves were analyzed to evaluate the diagnostic performance of the biomarker candidates.ResultsWe describe an effort to develop and analytically validate a multiplexed Tier 2 targeted PRM MS assay to quantify candidate ovarian cancer protein biomarkers in sera. Among the 64 peptides corresponding to 23 proteins in our PRM assay, 24 peptides corresponding to 16 proteins passed the assay validation acceptability criteria. A total of 6 of these peptides from insulin-like growth factor-binding protein 2 (IBP2), sex hormone-binding globulin (SHBG), and TIMP metalloproteinase inhibitor 1 (TIMP1) were quantified in sera from a cohort of 69 patients with early-stage HGSOC, late-stage HGSOC, benign ovarian conditions, and healthy (non-cancer) controls. Confirming the results from previously published studies using orthogonal analytical approaches, IBP2 was identified as a diagnostic biomarker candidate based on its significantly increased abundance in the late-stage HGSOC patient sera compared to the healthy controls and patients with benign ovarian conditions.ConclusionsA multiplexed targeted PRM MS assay was applied to detect candidate diagnostic biomarkers in HGSOC sera. To evaluate the clinical utility of the IBP2 PRM assay for HGSOC detection, further studies need to be performed using a larger patient cohort.

Quantitative phosphoproteomic analysis of chicken DF-1 cells infected with Eimeria tenella, using tandem mass tag (TMT) and parallel reaction monitoring (PRM) mass spectrometry.

Eimeria tenella is an obligate intracellular parasite which causes great harm to the poultry breeding industry. Protein phosphorylation plays a vital role in host cell-E. tenella interactions. However, no comprehensive phosphoproteomic analyses of host cells at various phases of E. tenella infection have been published. In this study, quantitative phosphoproteomic analysis of chicken embryo DF-1 fibroblasts that were uninfected (UI) or infected with E. tenella for 6h (PI6, the early invasion phase) or 36h (PI36, the trophozoite development phase) was conducted. A total of 10,122 phosphopeptides matched to 3,398 host cell phosphoproteins were identified and 13,437 phosphorylation sites were identified. Of these, 491, 1,253, and 275 differentially expressed phosphorylated proteins were identified in the PI6/UI, PI36/UI, and PI36/PI6 comparisons, respectively. KEGG pathway enrichment analysis showed that E. tenella modulated host cell processes through phosphorylation, including focal adhesion, regulation of the actin cytoskeleton, and FoxO signaling to support its early invasion phase, and modulating adherens junctions and the ErbB signaling pathway to favor its trophozoite development. These results enrich the data on the interaction between E. tenella and host cells and facilitate a better understanding of the molecular mechanisms underlying host-parasite relationships.

Use of volumetric absorptive microsampling and parallel reaction monitoring mass spectrometry for tacrolimus blood trough measurements at home in pediatric heart transplant patients

BackgroundMeasurement of trough levels for calcineurin inhibitors by venipuncture sampling is a mainstay of patient management in solid organ transplant recipients but challenging in pediatric patients. Volumetric Absorptive Microsampling (VAMS) is a patient-friendly, minimally invasive sampling technique to accurately collect blood. An assay for measurement of tacrolimus in blood using VAMS, coupled with parallel reaction monitoring (PRM) mass spectrometry, was validated in pediatric heart transplant patients. MethodsTacrolimus was measured by a newly developed high-resolution PRM assay and compared with low-resolution tandem mass spectrometry (MRM). Dried blood samples were collected from pediatric heart transplant patients (n = 35) using VAMS devices and a satisfaction survey was completed by patients/guardians. Tacrolimus concentrations were compared across whole liquid blood, dried blood spots, and capillary blood, and shipping stability determined. ResultsThe PRM assay was linear over a range 1–50 ng/mL, similar to MRM but had greater specificity due to reduced background noise. No significant differences in tacrolimus concentrations were observed between VAMS and venous blood. Tacrolimus dried on VAM tips was stable for 14 days and concentrations were unaffected by postal shipping. The variability in two simultaneously collected at-home patient samples was minimal – average concentration difference was 0.12 ± 0.94 ng/mL (p = 0.6) between paired samples. ConclusionA high resolution PRM mass spectrometry assay was developed for home-based dried blood collections for therapeutic monitoring of tacrolimus. The advantage of PRM was enhanced specificity and the VAMS devices provided a simple and convenient approach to blood sampling at home in pediatric heart transplant patients.

Synaptic Marker NPTX2 Predicts Risk of MCI Clinical Symptom Onset

AbstractBackgroundNeuronal pentraxin 2 (NPTX2) is a synaptic protein involved in the homeostatic regulation of cortical network dynamics and synaptic plasticity. Prior cross‐sectional studies suggest that NPTX2 levels decline across the spectrum of Alzheimer’s disease (AD), and that NPTX2 levels can distinguish individuals with normal cognition vs. mild cognitive impairment (MCI) and AD dementia. Less is known about how levels of NPTX2 among individuals with normal cognition relate to future clinical outcomes. To address this, we examined whether baseline levels of NPTX2 predicted time to onset of clinical symptoms of MCI, both alone and in combination with AD biomarkers, among individuals who were cognitively normal at baseline and have undergone 16.3y of follow‐up, on average.MethodCerebrospinal fluid (CSF) was collected at baseline from 269 cognitively normal BIOCARD Study participants (M baseline age = 57.7y), including 77 (29%) who subsequently progressed to MCI or dementia (Table 1). Baseline NPTX2 levels were measured from three highly correlated peptides using quantitative parallel reaction monitoring mass spectrometry. NPTX2 peptide levels were standardized and averaged to create an NPTX2 composite score. Baseline levels of Aβ42/Aβ40, p‐tau181, and t‐tau were measured using Lumipulse automated electrochemiluminescence assays.ResultIn Cox regression models, lower NPTX2 levels were significantly associated with earlier time to MCI clinical symptom onset (HR = 0.76, 95% CI (0.601, 0.962), p = 0.02; Figure 1a). After accounting for CSF AD biomarkers (i.e., Aβ42/Aβ40 and tau), the association between NPTX2 levels and MCI symptom onset appeared stronger (Table 2; Figure 1b). Importantly, NPTX2 did not interact with AD biomarker levels (all p > 0.11) or APOE‐ε4 genetic status (p = 0.71) in relation to MCI symptom onset.ConclusionCSF levels of the synaptic marker NPTX2 predict time to progress to MCI among individuals with normal cognition, suggesting that NPTX2 may be an early prognostic biomarker of synaptic dysfunction. Moreover, this association appears to be additive and independent of AD biomarker levels. Future work is underway to determine if NPTX2 provides resilience to cognitive impairment in the presence of AD pathology, and if there is a differential relationship with markers of amyloid vs. tau.

Quantitative Measurement of Transthyretin Mistargeting by Proximity Labeling and Parallel Reaction Monitoring.

Proximity labeling is a powerful approach for characterizing subcellular proteomes. We recently demonstrated that proximity labeling can be used to identify mistrafficking of secretory proteins, such as occurs during pre-emptive quality control (pre-QC) following endoplasmic reticulum (ER) stress. This assay depends on protein quantification by immunoblotting and densitometry, which sometimes suffers from poor sensitivity. Here, we integrate parallel reaction monitoring (PRM) mass spectrometry to enable a more quantitative platform, and assess how chemical ER stressors impact pre-QC of the model secretory protein transthyretin in HEK293T cells. We find that some drug treatments affect labeling efficiency, which can be controlled for by normalizing to APEX2 auto-labeling. While some chemical ER stress inducers including Brefeldin A and thapsigargin induce pre-QC, tunicamycin and dithiothreitol do not, indicating ER stress alone is not sufficient. This finding contrasts with the canonical model of pre-QC induction, and establishes the utility of our platform.

Proteomics-based analysis on the stress response mechanism of Bidens pilosa L. under cadmium exposure

Bidens pilosa L. (B. pilosa) has great potential for the phytoremediation of cadmium (Cd)-contaminated soils. However, the molecular mechanism underlying Cd tolerance and detoxification in B. pilosa is still unclear. In the present study, a 4D label-free quantification technique combined with liquid chromatography–parallel reaction monitoring mass spectrometry was used to explore the stress response mechanism of B. pilosa. Proteomic analysis revealed 213 and 319 differentially expressed proteins (DEPs) in the roots and leaves of B. pilosa, respectively, and 12 target proteins were selected for further analysis. SWISS-MODEL was used to predict the 3D structures of the target proteins. The cation-ATPase-N structural domain and an ATPase-E1-E2 motif, which help to regulate ATPase function, were detected in the TR10519_c0_g1_ORF protein. In addition, the TR6620_c0_g1_ORF_1 and TR611_c1_g1_ORF proteins contained peroxidase-1 and peroxidase-2 motifs. The TR11239_c0_g1_ORF protein was found to belong to the Fe-SOD family, to have a dimeric structure and to contain a relatively high proportion of α-helices but few β-sheets, which play important roles in reactive oxygen intermediate scavenging. Thus, the current study provides an overview of the proteomic response of B. pilosa in scavenging of Cd-induced reactive oxygen intermediates and reveals key proteins involved in the stress response of B. pilosa under Cd exposure.

Identification and verification of plasma protein biomarkers that accurately identify an ectopic pregnancy

BackgroundDifferentiating between a normal intrauterine pregnancy (IUP) and abnormal conditions including early pregnancy loss (EPL) or ectopic pregnancy (EP) is a major clinical challenge in early pregnancy. Currently, serial β-human chorionic gonadotropin (β-hCG) and progesterone are the most commonly used plasma biomarkers for evaluating pregnancy prognosis when ultrasound is inconclusive. However, neither biomarker can predict an EP with sufficient and reproducible accuracy. Hence, identification of new plasma biomarkers that can accurately diagnose EP would have great clinical value.MethodsPlasma was collected from a discovery cohort of 48 consenting women having an IUP, EPL, or EP. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by a label-free proteomics analysis to identify significant changes between pregnancy outcomes. A panel of 14 candidate biomarkers were then verified in an independent cohort of 74 women using absolute quantitation by targeted parallel reaction monitoring mass spectrometry (PRM-MS) which provided the capacity to distinguish between closely related protein isoforms. Logistic regression and Lasso feature selection were used to evaluate the performance of individual biomarkers and panels of multiple biomarkers to predict EP.ResultsA total of 1391 proteins were identified in an unbiased plasma proteome discovery. A number of significant changes (FDR ≤ 5%) were identified when comparing EP vs. non-EP (IUP + EPL). Next, 14 candidate biomarkers (ADAM12, CGA, CGB, ISM2, NOTUM, PAEP, PAPPA, PSG1, PSG2, PSG3, PSG9, PSG11, PSG6/9, and PSG8/1) were verified as being significantly different between EP and non-EP in an independent cohort (FDR ≤ 5%). Using logistic regression models, a risk score for EP was calculated for each subject, and four multiple biomarker logistic models were identified that performed similarly and had higher AUCs than models with single predictors.ConclusionsOverall, four multivariable logistic models were identified that had significantly better prediction of having EP than those logistic models with single biomarkers. Model 4 (NOTUM, PAEP, PAPPA, ADAM12) had the highest AUC (0.987) and accuracy (96%). However, because the models are statistically similar, all markers in the four models and other highly correlated markers should be considered in further validation studies.

Jianghu decoction and its active component polydatin inhibit inflammation and fibrotic lesions in the lungs of ILD mice via the AMPK signaling pathway

Ethnopharmacological relevanceRecently, interstitial lung disease (ILD) morbidity and mortality have been increasing with insidious epidemiological characteristics. Jianghu decoction (JH) is an effective Chinese medicine for ILD. Aim of the studyWe aimed to reveal the material basis and mechanism of action of JH in the treatment of ILD. Materials and methodsIn this study, an ILD mouse model was constructed with bleomycin. HE staining, transcriptome analysis, parallel reaction monitoring-mass spectrometry (PRM-MS), UPLC‒MS, and western blotting assays were conducted. ResultsHE staining results showed that JH effectively reduced inflammation and fibrosis foci in the lungs of the ILD model. Furthermore, transcriptome analysis revealed that JH regulates a set of biological signaling pathways related to immune inflammation and fibrosis. PRM-MS combined with western blotting was applied to detect inflammation and fibrosis involving proteins in lung tissue. JH effectively reversed the aberrant expression of HMGB1, RAGE, SEPTIN4, ACTA2, and ITGAV proteins in the model group. AMPK was identified as the core upstream regulatory protein for JH-mediated ILD regulation. In addition, UHPLC‒MS technology was applied to determine the active ingredients of JH. A total of 80 components were identified from JH, and polydatin (PD) was identified as the active ingredient that effectively alleviated lung fibrosis and inflammatory injury in ILD mice. To illustrate the molecular regulatory network of JH and PD in alleviating lung fibrosis and inflammatory injury, we also examined inflammation and fibrosis-related molecules downstream of the AMPK pathway with RT‒qPCR and western blotting. ConclusionsThe results showed that both JH and its active component PD exert synergistic inhibition on pulmonary fibrosis and inflammation. Specifically, the AMPK/PGC1α/PPARγ signaling pathway was activated, and the AMPK/HMGB1/RAGE signaling pathway was inhibited in ILD lungs responding to JH or PD administration.

NPTX2 in Cerebrospinal Fluid Predicts the Progression From Normal Cognition to Mild Cognitive Impairment.

This study examined whether cerebrospinal fluid (CSF) baseline levels of the synaptic protein NPTX2 predict time to onset of symptoms of mild cognitive impairment (MCI), both alone and when accounting for traditional CSF Alzheimer's disease (AD) biomarker levels. Longitudinal NPTX2 levels were also examined. CSF was collected longitudinally from 269 cognitively normal BIOCARD Study participants (mean baseline age = 57.7 years; mean follow-up = 16.3 years; n = 77 progressed to MCI/dementia). NPTX2 levels were measured from 3 correlated peptides using quantitative parallel reaction monitoring mass spectrometry. Levels of Aβ42 /Aβ40 , p-tau181 , and t-tau were measured from the same CSF specimens using Lumipulse automated electrochemiluminescence assays. In Cox regression models, lower baseline NPTX2 levels were associated with an earlier time to MCI symptom onset (hazard ratio [HR] = 0.76, SE = 0.09, p = 0.023). This association was significant for progression within 7 years (p = 0.036) and after 7 years from baseline (p = 0.001). Baseline NPTX2 levels improved prediction of time to MCI symptom onset after accounting for baseline AD biomarker levels (p < 0.01), and NPTX2 did not interact with the CSF AD biomarkers or APOE-ε4 genetic status. In linear mixed effects models, higher baseline p-tau181 and t-tau levels were associated with higher baseline levels of NPTX2 (both p < 0.001) and greater rates of NPTX2 declines over time. NPTX2 may be a valuable prognostic biomarker during preclinical AD that provides additive and independent prediction of MCI onset among individuals who are cognitively normal. We hypothesize that NPTX2-mediated circuit homeostasis confers resilience during the early phase of AD. ANN NEUROL 2023;94:620-631.

Orthogonal proteomics methods warrant the development of Duchenne muscular dystrophy biomarkers

BackgroundMolecular components in blood, such as proteins, are used as biomarkers to detect or predict disease states, guide clinical interventions and aid in the development of therapies. While multiplexing proteomics methods promote discovery of such biomarkers, their translation to clinical use is difficult due to the lack of substantial evidence regarding their reliability as quantifiable indicators of disease state or outcome. To overcome this challenge, a novel orthogonal strategy was developed and used to assess the reliability of biomarkers and analytically corroborate already identified serum biomarkers for Duchenne muscular dystrophy (DMD). DMD is a monogenic incurable disease characterized by progressive muscle damage that currently lacks reliable and specific disease monitoring tools.MethodsTwo technological platforms are used to detect and quantify the biomarkers in 72 longitudinally collected serum samples from DMD patients at 3 to 5 timepoints. Quantification of the biomarkers is achieved by detection of the same biomarker fragment either through interaction with validated antibodies in immuno-assays or through quantification of peptides by Parallel Reaction Monitoring Mass Spectrometry assay (PRM-MS).ResultsFive, out of ten biomarkers previously identified by affinity-based proteomics methods, were confirmed to be associated with DMD using the mass spectrometry-based method. Two biomarkers, carbonic anhydrase III and lactate dehydrogenase B, were quantified with two independent methods, sandwich immunoassays and PRM-MS, with Pearson correlations of 0.92 and 0.946 respectively. The median concentrations of CA3 and LDHB in DMD patients was elevated in comparison to those in healthy individuals by 35- and 3-fold, respectively. Levels of CA3 vary between 10.26 and 0.36 ng/ml in DMD patients whereas those of LDHB vary between 15.1 and 0.8 ng/ml.ConclusionsThese results demonstrate that orthogonal assays can be used to assess the analytical reliability of biomarker quantification assays, providing a means to facilitate the translation of biomarkers to clinical practice. This strategy also warrants the development of the most relevant biomarkers, markers that can be reliably quantified with different proteomics methods.

High-Sensitivity Detection toward SARS-CoV-2 S1 Glycoprotein by Parallel Reaction Monitoring Mass Spectrometry.

The outbreak of coronavirus disease 2019 (COVID-19) has overwhelmed the global economy and human well-being. On account of the sharp increase in test demand, there is a need for an accurate and alternative diagnosis method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, with the aim to specifically identify the trace SARS-CoV-2 S1 glycoprotein, we developed a high-sensitivity and high-selectivity diagnostic method based on the targeted parallel reaction monitoring (PRM) assay of eight selected peptides. This study emphasizes the outstanding detection sensitivity of 0.01 pg of the SARS-CoV-2 S1 glycoprotein even in the interference of other structural proteins, which to our knowledge is the current minimum limit of detection for the SARS-CoV-2 S1 glycoprotein. This technology could further identify 0.01 pg of the SARS-CoV-2 S1 glycoprotein in a spike pseudovirus, revealing its practical effectiveness. All our preliminary results throw light on the capability of the mass spectrometry-based targeted PRM assay to identify SARS-CoV-2 as a practicable orthogonal diagnostic tool. Furthermore, this technology could be extended to other pathogens (e.g., MERS-CoV S1 protein or SARS-CoV S1 protein) by quickly adjusting the targeted peptides of MS data acquisition. In summary, this strategy is universal and flexible and could be quickly adjusted to detect and discriminate different mutants and pathogens.

In-Depth Serum Proteomics Reveals the Trajectory of Hallmarks of Cancer in Hepatitis B Virus–Related Liver Diseases

Hepatocellular carcinoma (HCC) is a prevalent cancer in China, with chronic hepatitis B (CHB) and liver cirrhosis (LC) being high-risk factors for developing HCC. Here, we determined the serum proteomes (762 proteins) of 125 healthy controls and Hepatitis B virus–infected CHB, LC, and HCC patients and constructed the first cancerous trajectory of liver diseases. The results not only reveal that the majority of altered biological processes were involved in the hallmarks of cancer (inflammation, metastasis, metabolism, vasculature, and coagulation) but also identify potential therapeutic targets in cancerous pathways (i.e., IL17 signaling pathway). Notably, the biomarker panels for detecting HCC in CHB and LC high-risk populations were further developed using machine learning in two cohorts comprised of 200 samples (discovery cohort = 125 and validation cohort = 75). The protein signatures significantly improved the area under the receiver operating characteristic curve of HCC (CHB discovery and validation cohort = 0.953 and 0.891, respectively; LC discovery and validation cohort = 0.966 and 0.818, respectively) compared to using the traditional biomarker, alpha-fetoprotein, alone. Finally, selected biomarkers were validated with parallel reaction monitoring mass spectrometry in an additional cohort (n = 120). Altogether, our results provide fundamental insights into the continuous changes of cancer biology processes in liver diseases and identify candidate protein targets for early detection and intervention.

Abstract B015: Wildtype RAS activity and PI3K signaling as new vulnerabilities in cells with acquired resistance to sotorasib

Abstract Purpose: Sotorasib (AMG510) has demonstrated remarkable response in lung cancer patients with tumors driven by oncogenic KRASG12C mutation. However, recently published clinical data identified acquired mutations in RAS and other genomic alterations as potential mechanisms of resistance. The cause of resistance in more than half of the patient cohort was undetermined with genomic sequencing. We hypothesize that rewiring of signaling networks could be the newly acquired vulnerabilities in cells progressing after sotorasib treatment. Experimental Design: We performed whole exome sequencing, transcriptomic profiling, and mass spectrometry-based proteomics/phosphoproteomics of the parental and sotorasib resistant isogenic line of LU65 (LU65-AMGR). The omics analyses were integrated with functional screens to prioritize a set of druggable targets/pathways. Results: We did not observe obvious acquired mutations that drive resistance to sotorasib in LU65-AMGR cells. To investigate compensatory signaling critically important for the survival/growth of LU65-AMGR cells, we studied signaling perturbations using mass spectrometry-based phosphoproteomics approach. We identified 430 and 1,574 phosphosites differentially expressed in resistant cells (± 1.5-fold &amp; p &amp;lt; 0.05) with phosphotyrosine (pY) and global phosphoproteome (pS/T/Y) enrichment, respectively. Analysis of phosphoproteomics data identified increased phosphorylation of multiple RTKs including EGFR, HER2, HER3, IGF1R, AXL and PDGFRA, including others. RAS-GTP pull-down show increased levels of RAS-GTP and NRAS-GTP in the resistant cells. RAS isoform quantification using parallel reaction monitoring (MRM) mass spectrometry further confirmed increased activity of NRAS in pull-down samples. Transcriptomic analysis revealed elevated genes responsible for anti-apoptosis pathways mediated by PI3K/AKT signaling. Enrichment of transcription factors such as AP1 and AP-2A could drive enhanced EGF expression and EGFR phosphorylation in sotorasib resistant cells. Consistent with enhanced RTK and RAS activity, Western blots confirmed higher phosphorylation of ERK and AKT in LU65-AMGR cells. We show higher EGFR phosphorylation in resistant cells when compared to the parental counterpart. While LU65 cells viability is reduced markedly by sotorasib combination with afatinib, an irreversible HER family inhibitor, resistant cells showed lesser effect on cell viability with afatinib combination. Consistent with our in vitro observations, the sotorasib and afatinib combination treatment significantly regressed tumor growth only in LU65 xenografts, but not in LU65-AMGR xenografts. Conclusions: Our data suggest that activation of multiple RTKs maintains RAS activity and PI3K signaling in LU65-AMGR cells. Thus, dual pan-RAS and PI3K inhibition could serve as promising strategy of treatment in relapsed tumors identified with WT RAS and PI3K signaling activation. Citation Format: Denis Imbody, Hitendra S. Solanki, Bina Desai, Paul A. Stewart, Yaakov Stern, Ryoji Kato, Anurima Majumder, Liznair Bridenstine, Aobuli Xieraili, Bin Fang, Lancia Darville, Fumi Kinose, John M. Koomen, Uwe Rix, Andriy Marusyk, Eric B. Haura. Wildtype RAS activity and PI3K signaling as new vulnerabilities in cells with acquired resistance to sotorasib [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B015.

Placental Remote Control of Fetal Metabolism: Trophoblast mTOR Signaling Regulates Liver IGFBP-1 Phosphorylation and IGF-1 Bioavailability.

The mechanisms mediating the restricted growth in intrauterine growth restriction (IUGR) remain to be fully established. Mechanistic target of rapamycin (mTOR) signaling functions as a placental nutrient sensor, indirectly influencing fetal growth by regulating placental function. Increased secretion and the phosphorylation of fetal liver IGFBP-1 are known to markedly decrease the bioavailability of IGF-1, a major fetal growth factor. We hypothesized that an inhibition of trophoblast mTOR increases liver IGFBP-1 secretion and phosphorylation. We collected conditioned media (CM) from cultured primary human trophoblast (PHT) cells with a silenced RAPTOR (specific inhibition of mTOR Complex 1), RICTOR (inhibition of mTOR Complex 2), or DEPTOR (activates both mTOR Complexes). Subsequently, HepG2 cells, a well-established model for human fetal hepatocytes, were cultured in CM from PHT cells, and IGFBP-1 secretion and phosphorylation were determined. CM from PHT cells with either mTORC1 or mTORC2 inhibition caused the marked hyperphosphorylation of IGFBP-1 in HepG2 cells as determined by 2D-immunoblotting while Parallel Reaction Monitoring-Mass Spectrometry (PRM-MS) identified increased dually phosphorylated Ser169 + Ser174. Furthermore, using the same samples, PRM-MS identified multiple CK2 peptides coimmunoprecipitated with IGFBP-1 and greater CK2 autophosphorylation, indicating the activation of CK2, a key enzyme mediating IGFBP-1 phosphorylation. Increased IGFBP-1 phosphorylation inhibited IGF-1 function, as determined by the reduced IGF-1R autophosphorylation. Conversely, CM from PHT cells with mTOR activation decreased IGFBP-1 phosphorylation. CM from non-trophoblast cells with mTORC1 or mTORC2 inhibition had no effect on HepG2 IGFBP-1 phosphorylation. Placental mTOR signaling may regulate fetal growth by the remote control of fetal liver IGFBP-1 phosphorylation.

Scutellarin Alleviates Ischemic Brain Injury in the Acute Phase by Affecting the Activity of Neurotransmitters in Neurons.

Cerebral ischemic stroke is a common neuron loss disease that is caused by the interruption of the blood supply to the brain. In order to enhance the CIS outcome, both identifying the treatment target of ischemic brain damage in the acute phase and developing effective therapies are urgently needed. Scutellarin had been found to be beneficial to ischemic injuries and has been shown to have potent effects in clinical application on both stroke and myocardial infarction. However, whether scutellarin improves ischemic brain damage in the acute phase remains unknown. In this study, the protective effects of scutellarin on ischemic brain damage in the acute phase (within 12 h) were illustrated. In middle cerebral artery occlusion and reperfusion (MCAO/R) modeling rats, the Z-Longa score was significantly down-regulated by 25% and 23.1%, and the brain infarct size was reduced by 26.95 ± 0.03% and 25.63 ± 0.02% when responding to high-dose and low-dose scutellarin treatments, respectively. H&E and TUNEL staining results indicated that the neuron loss of the ischemic region was improved under scutellarin treatment. In order to investigate the mechanism of scutellarin's effects on ischemic brain damage in the acute phase, changes in proteins and metabolites were analyzed. The suppression of scutellarin on the glutamate-inducing excitatory amino acid toxicity was strongly indicated in the study of both proteomics and metabolomics. A molecular docking experiment presented strong interactions between scutellarin and glutamate receptors, which score much higher than those of memantine. Further, by performing a parallel reaction monitoring-mass spectrometry (PRM-MS) study on both the cortex and hippocampus tissue of the ischemic region, we screened the scutellarin-regulating molecules that are involved in both the release and transportation of neurotransmitters. It was found that the aberrant levels of glutamate receptors, including EAAT2, GRIN1, GRIN2B, and GRM1, as well as of other glutamatergic pathway-involving proteins, including CAMKK2, PSD95, and nNOS, were significantly regulated in the ischemic cortex. In the hippocampus, EAAT2, GRIN1, nNOS, and CAM were significantly regulated. Taken together, scutellarin exerts potent effects on ischemic brain damage in the acute phase by regulating the activity of neurotransmitters and reducing the toxicity of excitatory amino acids in in neurons.

Endo-lysosomal protein concentrations in CSF from patients with frontotemporal dementia caused by CHMP2B mutation.

Increasing evidence implicates proteostatic dysfunction as an early event in the development of frontotemporal dementia (FTD). This study aimed to explore potential cerebrospinal fluid (CSF) biomarkers associated with the proteolytic systems in genetic FTD caused by CHMP2B mutation. Combining solid-phase extraction and parallel reaction monitoring mass spectrometry, a panel of 47 peptides derived from 20 proteins was analyzed in CSF from 31 members of the Danish CHMP2B-FTD family. Compared with family controls, mutation carriers had significantly higher levels of complement C9, lysozyme and transcobalamin II, and lower levels of ubiquitin, cathepsin B, and amyloid precursor protein. Lower CSF ubiquitin concentrations in CHMP2B mutation carriers indicate that ubiquitin levels relate to the specific disease pathology, rather than all-cause neurodegeneration. Increased lysozyme and complement proteins may indicate innate immune activation. Altered levels of amyloid precursor protein and cathepsins have previously been associated with impaired lysosomal proteolysis in FTD. CSF markers of proteostasis were explored in CHMP2B-mediated frontotemporal dementia (FTD).31 members of the Danish CHMP2B-FTD family were included.We used solid-phase extraction and parallel reaction monitoring mass spectrometry.Six protein levels were significantly altered in CHMP2B-FTD compared with controls.Lower CSF ubiquitin levels in patients suggest association with disease mechanisms.