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Personalized cancer immunotherapies such as therapeutic vaccines and adoptive transfer of T cell receptor-transgenic T cells rely on the presentation of tumor-specific peptides by human leukocyte antigen class I molecules to cytotoxic T cells. Such neoepitopes can for example arise from somatic mutations and their identification is crucial for the rational design of new therapeutic interventions. Liquid chromatography mass spectrometry (LC-MS)-based immunopeptidomics is the only method to directly prove actual peptide presentation and we have developed a parameter optimization workflow to tune targeted assays for maximum detection sensitivity on a per peptide basis, termed optiPRM. Optimization of collision energy using optiPRM allows for the improved detection of low abundant peptides that are very hard to detect using standard parameters. Applying this to immunopeptidomics, we detected a neoepitope in a patient-derived xenograft from as little as 2.5 × 106 cells input. Application of the workflow on small patient tumor samples allowed for the detection of five mutation-derived neoepitopes in three patients. One neoepitope was confirmed to be recognized by patient T cells. In conclusion, optiPRM, a targeted MS workflow reaching ultra-high sensitivity by per peptide parameter optimization, makes the identification of actionable neoepitopes possible from sample sizes usually available in the clinic.

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The creatine-phosphocreatine cycle serves as a crucial temporary energy buffering system in the brain, regulated by brain creatine kinase (CKB), in maintaining Adenosine triphosphate (ATP) levels. Alzheimer's disease (AD) has been linked to increased CKB oxidation and loss of its regulatory function, although specific pathological processes and affected cell types remain unclear. In our study, cerebral cortex samples from individuals with AD, dementia with Lewy bodies (DLB), and age-matched controls were analyzed using antibody-based methods to quantify CKB levels and assess alterations associated with disease processes. Two independently validated antibodies exclusively labeled astrocytes in the human cerebral cortex. Combining immunofluorescence (IF) and mass spectrometry (MS), we explored CKB availability in AD and DLB cases. IF and Western blot analysis demonstrated a loss of CKB immunoreactivity correlated with increased plaque load, severity of tau pathology, and Lewy body pathology. However, transcriptomics data and targeted MS demonstrated unaltered total CKB levels, suggesting posttranslational modifications (PTMs) affecting antibody binding. This aligns with altered efficiency at proteolytic cleavage sites indicated in the targeted MS experiment. These findings highlight that the proper function of astrocytes, understudied in the brain compared with neurons, is highly affected by PTMs. Reduction in ATP levels within astrocytes can disrupt ATP-dependent processes, such as the glutamate-glutamine cycle. As CKB and the creatine-phosphocreatine cycle are important in securing constant ATP availability, PTMs in CKB, and astrocyte dysfunction may disturb homeostasis, driving excitotoxicity in the AD brain. CKB and its activity could be promising biomarkers for monitoring early-stage energy deficits in AD.

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DNA methylation in insects is generally low in abundance, and its role is not well understood. It is often localised in protein coding regions and associated with the expression of 'housekeeping' genes. Few studies have explored DNA methylation dynamics during lifecycle stage transitions in holometabolous (metamorphosing) insects. Using targeted mass spectrometry, we have found a significant difference in global DNA methylation levels between larvae, pupae and adults of Helicoverpa armigera (Lepidoptera: Noctuidae) Hübner, a polyphagous pest of agricultural importance. Whole-genome bisulfite sequencing confirmed these observations and pointed to non-CG context being the primary explanation for the difference observed between pupa and adult. Non-CG methylation was enriched in genes specific to various signalling pathways (Hippo signalling, Hedgehog signalling and mitogen-activated protein kinase (MAPK) signalling) and ATP-dependent chromatin remodelling. Understanding the function of this epigenetic mark could be a target in future studies focusing on integrated pest management.

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Targeted mass spectrometry (MS)-based absolute quantitative analysis has been increasingly used in biomarker discovery. The ability to accurately measure the masses by MS enabled the use of isotope-incorporated surrogates having virtually identical physiochemical properties with the target analytes as calibrators. Such a unique capacity allowed for accurate in-sample calibration. Current in-sample calibration uses multiple isotopologues or structural analogues for both the surrogate and the internal standard. Here, we simplified this common practice by using endogenous light peptides as the internal standards and used a mathematical deduction of "heavy matching light, HML" to directly quantify an endogenous analyte. This method provides all necessary assay performance parameters in the authentic matrix, including the lower limit of quantitation (LLOQ) and intercept of the calibration curve, by using only a single isotopologue of the analyte. This method can be applied to the quantitation of proteins, peptides, and small molecules. Using this method, we quantified the efficiency of heart tissue digestion and recovery using sodium deoxycholate as a detergent and two spiked exogenous proteins as mimics of heart proteins. The results demonstrated the robustness of the assay.

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Picornaviridae represent a large family of single-stranded positive RNA viruses of which different members can infect both humans and animals. These include the enteroviruses (e.g., poliovirus, coxsackievirus, and rhinoviruses) as well as the cardioviruses (e.g., encephalomyocarditis virus). Picornaviruses have evolved to interact with, use, and/or evade cellular host systems to create the optimal environment for replication and spreading. It is known that viruses modify kinase activity during infection, but a proteome-wide overview of the (de)regulation of cellular kinases during picornavirus infection is lacking. To study the kinase activity landscape during picornavirus infection, we here applied dedicated targeted mass spectrometry-based assays covering ∼40% of the human kinome. Our data show that upon infection, kinases of the MAPK pathways become activated (e.g., ERK1/2, RSK1/2, JNK1/2/3, and p38), while kinases involved in regulating the cell cycle (e.g., CDK1/2, GWL, and DYRK3) become inactivated. Additionally, we observed the activation of CHK2, an important kinase involved in the DNA damage response. Using pharmacological kinase inhibitors, we demonstrate that several of these activated kinases are essential for the replication of encephalomyocarditis virus. Altogether, the data provide a quantitative understanding of the regulation of kinome activity induced by picornavirus infection, providing a resource important for developing novel antiviral therapeutic interventions.

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Mass spectrometry (MS) assays offer exceptional capabilities in high multiplexity, specificity, and throughput. As proteomics technologies continue advancements to identify new disease biomarkers, transition of these innovations from research settings to clinical applications becomes imperative. To meet the rigorous regulatory standards of clinical laboratories, development of a clinical protein MS assay necessitates adherence to stringent criteria. To illustrate the process, this project focused on using thyroglobulin (Tg) as a biomarker and an immuno-multiple reaction monitoring (iMRM) MS-based assay as a model for establishing a Clinical Laboratory Improvement Amendments (CLIA) compliant laboratory within the Centers of Genomic and Precision Medicine, National Taiwan University. The chosen example also illustrates the clinical utility of MS assays to complement conventional immunoassay-based methods, particularly in cases where the presence of autoantibodies in 10-30% of patients hinders accuracy. The laboratory design entails a comprehensive coordination in spatial layout, workflow organization, equipment selection, ventilation systems, plumbing, electrical infrastructure, documentation procedures, and communication protocols. Practical aspects of the transformation process, including preparing laboratory facilities, testing environments, instrument validation, assay development and validation, quality management, sample testing, and personnel competency, are discussed. Finally, concordant results in proficiency testing demonstrate the harmonization with the University of Washington Medical Center and the quality assurance of the CLIA-equivalent Tg-iMRM MS assay established in Taiwan. The realization of this model protein MS assay in Taiwan highlights the feasibility of international joint development and provides a detailed reference map to expedite the implementation of more MS-based protein assays in clinical laboratories for patient care.

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BACKGROUND: Ovarian 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. METHODS: To 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. RESULTS: We 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. CONCLUSIONS: A 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.

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Alzheimer's disease (AD) is characterized by several neuropathological changes, mainly extracellular amyloid aggregates (plaques), intraneuronal inclusions of phosphorylated tau (tangles), as well as neuronal and synaptic degeneration, accompanied by tissue reactions to these processes (astrocytosis and microglial activation) that precede neuronal network disturbances in the symptomatic phase of the disease. A number of biomarkers for these brain tissue changes have been developed, mainly using immunoassays. In this review, we discuss how targeted mass spectrometry (TMS) can be used to validate and further characterize classes of biomarkers reflecting different AD pathologies, such as tau- and amyloid-beta pathologies, synaptic dysfunction, lysosomal dysregulation, and axonal damage, and the prospect of using TMS to measure these proteins in clinical research and diagnosis. TMS advantages and disadvantages in relation to immunoassays are discussed, and complementary aspects of the technologies are discussed.

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OBJECTIVES: Minimal residual disease status in multiple myeloma is an important prognostic biomarker. Recently, personalized blood-based targeted mass spectrometry (MS-MRD) was shown to provide a sensitive and minimally invasive alternative to measure minimal residual disease. However, quantification of MS-MRD requires a unique calibrator for each patient. The use of patient-specific stable isotope labelled (SIL) peptides is relatively costly and time-consuming, thus hindering clinical implementation. Here, we introduce a simplification of MS-MRD by using an off-the-shelf calibrator. METHODS: SILuMAB-based MS-MRD was performed by spiking a monoclonal stable isotope labeled IgG, SILuMAB-K1, in the patient serum. The abundance of both M-protein-specific peptides and SILuMAB-specific peptides were monitored by mass spectrometry. The relative ratio between M-protein peptides and SILuMAB peptides allowed for M-protein quantification. We assessed linearity, sensitivity and reproducibility of SILuMAB-based MS-MRD in longitudinally collected sera from the IFM-2009 clinical trial. RESULTS: A linear dynamic range was achieved of over 5 log scales, allowing for M-protein quantification down to 0.001 g/L. The inter-assay CV of SILuMAB-based MS-MRD was on average 11 %. Excellent concordance between SIL- and SILuMAB-based MS-MRD was shown (R2>0.985). Additionally, signal intensity of spiked SILuMAB can be used for quality control purpose to assess system performance and incomplete SILuMAB digestion can be used as quality control for sample preparation. CONCLUSIONS: Compared to SIL peptides, SILuMAB-based MS-MRD improves the reproducibility, turn-around-times and cost-efficacy of MS-MRD without diminishing its sensitivity and specificity. Furthermore, SILuMAB can be used as a MS-MRD quality control tool to monitor sample preparation efficacy and assay performance.

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Streck tubes are commonly used to collect blood samples to preserve cell-free circulating DNA. They contain imidazolidinyl urea as a formaldehyde-releasing agent to stabilize cells. We investigated whether the released formaldehyde leads to crosslinking of intracellular proteins. Therefore, we employed a shotgun proteomics experiment on human peripheral blood mononuclear cells (PBMCs) that were isolated from blood collected in Streck tubes, EDTA tubes, EDTA tubes containing formaldehyde, or EDTA tubes containing allantoin. The identified crosslinks were validated in parallel reaction monitoring LC/MS experiments. In total, we identified and validated 45 formaldehyde crosslinks in PBMCs from Streck tubes, which were also found in PBMCs from formaldehyde-treated blood, but not in EDTA- or allantoin-treated samples. Most were derived from cytoskeletal proteins and histones, indicating the ability of Streck tubes to fix cells. In addition, we confirm a previous observation that formaldehyde crosslinking of proteins induces a +24 Da mass shift more frequently than a +12 Da shift. The crosslinking capacity of Streck tubes needs to be considered when selecting blood-collection tubes for mass-spectrometry-based proteomics or metabolomic experiments.

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INTRODUCTION: Clinical proteomics studies of Alzheimer's disease (AD) research aim to identify biomarkers useful for clinical research, diagnostics, and improve our understanding of the pathological processes involved in the disease. The rapidly increasing performance of proteomics technologies is likely to have great impact on AD research. AREAS COVERED: We review recent proteomics approaches that have advanced the field of clinical AD research. Specifically, we discuss the application of targeted mass spectrometry (MS), labeling-based and label-free MS-based as well as affinity-based proteomics to AD biomarker development, underpinning their importance with the latest impactful clinical studies. We evaluate how proteomics technologies have been adapted to meet current challenges. Finally, we discuss the limitations and potential of proteomics techniques and whether their scope might extend beyond current research-based applications. EXPERT OPINION: To date, proteomics technologies in the AD field have been largely limited to AD biomarker discovery. The recent development of the first successful disease-modifying treatments of AD will further increase the need for blood biomarkers for early, accurate diagnosis, and CSF biomarkers that reflect specific pathological processes. Proteomics has the potential to meet these requirements and to progress into clinical routine practice, provided that current limitations are overcome.

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Targeted mass spectrometry (MS)-based proteomic assays, such as multiplexed multiple reaction monitoring (MRM)-MS assays, enable sensitive and specific quantification of proteotypic peptides as stoichiometric surrogates for proteins. Efforts are underway to expand the use of MRM-MS assays in clinical environments, which requires a reliable strategy to monitor proteolytic digestion efficiency within individual samples. Towards this goal, extended stable isotope-labeled standard (SIS) peptides (hE), which incorporate native proteolytic cleavage sites, can be spiked into protein lysates prior to proteolytic (trypsin) digestion, and release of the tryptic SIS peptide (hT) can be monitored. However, hT measurements alone cannot monitor the extent of digestion and may be confounded by matrix effects specific to individual patient samples; therefore, they are not sufficient to monitor sample-to-sample digestion variability. We hypothesized that measuring undigested hE, along with its paired hT, would improve detection of digestion issues compared to only measuring hT. We tested the ratio of the SIS pair measurements, or hE/hT, as a quality control (QC) metric of trypsin digestion for two MRM assays: a direct-MRM (398 targets) and an immuno-MRM (126 targets requiring immunoaffinity peptide enrichment) assay, with extended SIS peptides observable for 54% (216) and 62% (78) of the targets, respectively. We evaluated the quantitative bias for each target in a series of experiments that adversely affected proteolytic digestion (e.g., variable digestion times, pH, and temperature). We identified a subset of SIS pairs (36 for the direct-MRM, 7 for the immuno-MRM assay) for which the hE/hT ratio reliably detected inefficient digestion that resulted in decreased assay sensitivity and unreliable endogenous quantification. The hE/hT ratio was more responsive to a decrease in digestion efficiency than a metric based on hT measurements alone. For clinical-grade MRM-MS assays, this study describes a ready-to-use QC panel and also provides a road map for designing custom QC panels.

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Alzheimer's disease (AD), a neurodegenerative disorder, is the most common cause of dementia in the elderly population. Since its original description, there has been intense debate regarding the factors that trigger its pathology. It is becoming apparent that AD is more than a brain disease and harms the whole-body metabolism. We analyzed 630 polar and apolar metabolites in the blood of 20 patients with AD and 20 healthy individuals, to determine whether the composition of plasma metabolites could offer additional indicators to evaluate any alterations in the metabolic pathways related to the illness. Multivariate statistical analysis showed that there were at least 25 significantly dysregulated metabolites in patients with AD compared with the controls. Two membrane lipid components, glycerophospholipids and ceramide, were upregulated, whereas glutamic acid, other phospholipids, and sphingolipids were downregulated. The data were analyzed using metabolite set enrichment analysis and pathway analysis using the KEGG library. The results showed that at least five pathways involved in the metabolism of polar compounds were dysregulated in patients with AD. Conversely, the lipid pathways did not show significant alterations. These results support the possibility of using metabolome analysis to understand alterations in the metabolic pathways related to AD pathophysiology.

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Per- and polyfluoroalkyl substances (PFAS) are a group of manufactured chemicals that are resistant to degradation and thus persistent in the environment. The presence, uptake, and accumulation of PFAS is dependent upon the physiochemical properties of the PFAS and matrix, as well as the environmental conditions since the time of release. The objective of this study was to measure the extent of PFAS contamination in surface water and sediment from nine vulnerable aquatic systems throughout Florida. PFAS were detected at all sampling locations with sediment exhibiting greater PFAS concentrations when compared to surface water. At most locations, elevated concentrations of PFAS were identified around areas of increased human activity, such as airports, military bases, and wastewater effluents. The results from the present study highlight the ubiquitous presence of PFAS in vital Florida waterways and filled an important gap in understanding the distribution of PFAS in dynamic, yet vulnerable, aquatic environments.

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BACKGROUND: Non-cholesterol sterols, as well as plant sterols, cross the blood-brain barrier and, thus, can be incorporated into cell membranes, affecting the cell's inflammatory response. The aim of our work was to develop an analytical protocol for a quantitative assessment of the sterol composition within the membrane microdomains of microglia. METHODS: A protocol for cell membrane isolation using OptiPrepTM gradient ultracentrifugation, in combination with a targeted mass spectrometry (LC-MS/MS)-based assay, was developed and validated for the quantitative analysis of free sterols in microglia cell membranes. RESULTS: Utilizing an established LC-MS/MS assay, cholesterol and seven non-cholesterol sterols were analyzed with a limit of detection from 0.001 to 0.05 mg/L. Applying the detergent-free isolation of SIM-A9 microglia cell membranes, cholesterol (CH), desmosterol (DE), lanosterol (LA) stigmasterol (ST), beta-sitosterol (SI) and campesterol (CA) were quantified with coefficients of variations between 6 and 29% (fractions 4-6, n = 5). The highest concentrations of non-CH sterols within the microglia plasma membranes were found in the microdomain region (DE>LA>SI>ST>CA), with ratios to CH ranging from 2.3 to 435 lower abundancies. CONCLUSION: By applying our newly developed and validated analytical protocol, we show that the non-CH sterol concentration is about 38% of the total sterol content in microglia membrane microdomains. Further investigations must clarify how changes in the non-sterol composition influence membrane fluidity and cell signaling.

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Immunoaffinity mass spectrometry as an approach for diagnostic biomarker assays combines the advantages of antibody selectivity with the multiplexing and analytical performance of mass spectrometry. A method has been developed to detect and quantify three protein biomarkers for a diabetic kidney disease prognostic assay, PromarkerD. The methodology reflects an immunoaffinity approach compatible with higher throughput and robust clinical application. After preparation and purification of antibody-bead conjugates for the three target proteins, an immunoaffinity capture step provides a solution for reduction, alkylation, and digestion on-bead. Targeted mass spectrometry provides a quantitative measure of each biomarker in a rapid 8 min run using a microflow LCMS workflow.

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Targeted mass spectrometry using multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM) has been commonly used for protein biomarker validation in plasma, serum, or other clinically relevant specimens due to its high specificity, selectivity, and multiplexing capability compared with immunoassays. As the emerging mode termed parallel accumulation-serial fragmentation (prmPASEF) significantly improved analyte throughput (100-1000), sensitivity (attomole level), and acquisition speed, it promises to broaden the application of targeted mass spectrometry to simultaneous biomarker discovery and validation with high accuracy. Here, we summarize the general approach of the MRM and PRM techniques used for serum/plasma proteomics and describe a detailed step-by-step procedure for the development of MRM/PRM assays for secreted proteins.

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A meta-analysis of the results of targeted quantitative screening of human blood plasma was performed to generate a reference standard kit that can be used for health analytics. The panel included 53 of the 296 proteins that form a "stable" part of the proteome of a healthy individual; these proteins were found in at least 70% of samples and were characterized by an interindividual coefficient of variation <40%. The concentration range of the selected proteins was 10−10−10−3 M and enrichment analysis revealed their association with rare familial diseases. The concentration of ceruloplasmin was reduced by approximately three orders of magnitude in patients with neurological disorders compared to healthy volunteers, and those of gelsolin isoform 1 and complement factor H were abruptly reduced in patients with lung adenocarcinoma. Absolute quantitative data of the individual proteome of a healthy and diseased individual can be used as the basis for personalized medicine and health monitoring. Storage over time allows us to identify individual biomarkers in the molecular landscape and prevent pathological conditions.

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Prostate cancer (PCa) is the second leading cause of male cancer-related deaths in the United States. The pre-mature forms of prostate-specific antigen (PSA), proPSA, were shown to be associated with PCa. However, there is a technical challenge in the development of antibody-based immunoassays for specific recognition of each individual proPSA isoform. Herein, we report the development of highly specific, antibody-free, targeted mass spectrometry assays for simultaneous quantification of [-2], [-4], [-5], and [-7] proPSA isoforms in voided urine. The newly developed proPSA assays capitalize on Lys-C digestion to generate surrogate peptides with appropriate length (9-16 amino acids) along with long-gradient liquid chromatography separation. The assay utility of these isoform markers was evaluated in a cohort of 30 well-established clinical urine samples for distinguishing PCa patients from healthy controls. Under the 95% confidence interval, the combination of [-2] and [-4] proPSA isoforms yields the area under curve (AUC) of 0.86, and the AUC value for the combined all four isoforms was calculated to be 0.85. We have further verified [-2]proPSA, the dominant isoform, in an independent cohort of 34 clinical urine samples. Validation of proPSA isoforms in large-scale cohorts is needed to demonstrate their potential clinical utility.

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Atherosclerotic cardiovascular disease is the leading cause of death worldwide. For decades, mouse modeling of atherosclerosis has been the mainstay for preclinical testing of genetic and pharmacological intervention. Mouse models of atherosclerosis depend on supraphysiological levels of circulating cholesterol carried in lipoprotein particles. Lipoprotein particles vary in atherogenicity, and it is critical to monitor lipoprotein levels during preclinical interventions in mice. Unfortunately, the small plasma volumes typically harvested during preclinical experiments limit analyses to measuring total cholesterol and triglyceride levels. Here we developed a high-throughput, low-cost targeted multiple reaction monitoring (MRM) stable isotope dilution (SID) mass spectrometry assay for simultaneous relative quantification of nine apolipoproteins using a few microliters of mouse plasma. We applied the MRM assay to investigate the plasma apolipoproteome of two atherosclerosis models: the widely used ApoE knockout model and the emerging recombinant adeno-associated virus-mediated hepatic Pcsk9 overexpression model. By applying the assay on size-exclusion chromatography-separated plasma pools, we provide in-depth characterization of apolipoprotein distribution across lipoprotein species in these models, and finally, we use the assay to quantify apolipoprotein deposition in mouse atherosclerotic plaques. Taken together, we report development and application of an MRM assay that can be adopted by fellow researchers to monitor the mouse plasma apolipoproteome during preclinical investigations.

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