High-resolution Parallel Reaction Monitoring Research Articles

Brevican and Neurocan Peptides as Potential Cerebrospinal Fluid Biomarkers for Differentiation Between Vascular Dementia and Alzheimer's Disease.

Brevican and neurocan are central nervous system-specific extracellular matrix proteoglycans. They are degraded by extracellular enzymes, such as metalloproteinases. However, their degradation profile is largely unexplored in cerebrospinal fluid (CSF). The study aim was to quantify proteolytic peptides derived from brevican and neurocan in human CSF of patients with Alzheimer's disease (AD) and vascular dementia (VaD) compared with controls. The first cohort consisted of 75 individuals including 25 patients with AD, 7 with mild cognitive impairment (MCI) diagnosed with AD upon follow-up, 10 patients with VaD or MCI diagnosed with VaD upon follow-up, and 33 healthy controls and cognitively stable MCI patients. In the second cohort, 31 individuals were included (5 AD patients, 14 VaD patients and 12 healthy controls). Twenty proteolytic peptides derived from brevican (n = 9) and neurocan (n = 11) were quantified using high-resolution parallel reaction monitoring mass spectrometry. In the first cohort, the majority of CSF concentrations of brevican and neurocan peptides were significantly decreased inVaDas compared withADpatients (AUC=0.83.0.93, p≤0.05) and as compared with the control group (AUC=0.79.0.87, p≤0.05). In the second cohort, CSF concentrations of two brevican peptides (B87, B156) were significantly decreased in VaD compared with AD (AUC=0.86.0.91, p≤0.05) and to controls (AUC=0.80.0.82, p≤0.05), while other brevican and neurocan peptides showed a clear trend to be decreased in VaD compared with AD (AUC=0.64.80, p>0.05). No peptides differed between AD and controls. Brevican and neurocan peptides are potential diagnostic biomarkers for VaD, with ability to separate VaD from AD.

Quantification of total apolipoprotein E and its isoforms in cerebrospinal fluid from patients with neurodegenerative diseases

BackgroundThe human APOE gene, which codes for apolipoprotein E (apoE), has three major polymorphic alleles: ε2, ε3, and ε4 that give rise to amino acid substitutions. APOE-ε4 is a strong risk factor of sporadic Alzheimer’s disease (AD) but the reason why is still unknown despite intense research for more than 20 years. The aim of the study was to investigate if the concentrations of total apoE and the specific apoE isoforms in cerebrospinal fluid (CSF) differ between various neurodegenerative diseases and control individuals, as well as among the APOE genotypes.MethodsQuantification of total apoE and specific apoE isoforms (E2, E3, and E4) in CSF was performed using high-resolution parallel reaction monitoring mass spectrometry. In total, 1820 individuals were involved in the study including clinically diagnosed AD patients (n = 228), cognitively unimpaired (CU) patients (n = 896), and patients with other neurodegenerative disorders (n = 696). Follow-up data was available for 100 individuals, assessed at two time points. Subjects were dichotomized based on an Aβ42/40 CSF concentration ratio cut-off into Aβ positive (Aβ+, < 0.091) and Aβ negative (Aβ−, > 0.091) groups.ResultsEven though there was a significant increase of total apoE in the amyloid β-positive (Aβ+) group compared with amyloid β-negative (Aβ−) individuals (p < 0.001), the magnitude of the effect was very small (AUC = 0.55). Moreover, CSF total apoE concentrations did not differ between Aβ− CU controls and clinically diagnosed AD patients. There was a difference in concentration between isoforms in heterozygous individuals in an isoform-dependent manner (E2 < E3 < E4) (p < 0.001, AUC = 0.64–0.69), and these associations remained when dichotomizing the samples into Aβ+ and Aβ− groups (p < 0.01, AUC = 0.63–0.74). In the cohort with follow-up samples, neither total apoE nor isoform-specific apoE concentrations differed between the two time points (p > 0.05).ConclusionsThe results indicate that neither the concentrations of total apoE nor the different apoE isoforms in CSF are associated with APOE-ε4 carrier status, Aβ status, or clinical dementia diagnoses.

Application of Stable Isotope Tracing to Elucidate Metabolic Dynamics During Yarrowia lipolytica α-Ionone Fermentation.

SummaryTargeted metabolite analysis in combination with 13C-tracing is a convenient strategy to determine pathway activity in biological systems; however, metabolite analysis is limited by challenges in separating and detecting pathway intermediates with current chromatographic methods. Here, a hydrophilic interaction chromatography tandem mass spectrometry approach was developed for improved metabolite separation, isotopologue analysis, and quantification. The physiological responses of a Yarrowia lipolytica strain engineered to produce ∼400 mg/L α-ionone and temporal changes in metabolism were quantified (e.g., mevalonate secretion, then uptake) indicating bottleneck shifts in the engineered pathway over the course of fermentation. Dynamic labeling results indicated limited tricarboxylic acid cycle label incorporation and, combined with a measurable ATP shortage during the high ionone production phase, suggested that electron transport and oxidative phosphorylation may limit energy supply and strain performance. The results provide insights into terpenoid pathway metabolic dynamics of non-model yeasts and offer guidelines for sensor development and modular engineering.

Pharmacokinetic Analysis of a Novel Human EGFRvIII:CD3 Bispecific Antibody in Plasma and Whole Blood Using a High-Resolution Targeted Mass Spectrometry Approach.

Bispecific single chain antibody fragments (bi-scFv) represent an emerging class of biotherapeutics. We recently developed a fully human bi-scFv (EGFRvIII:CD3 bi-scFv) with the goal of redirecting CD3-expressing T cells to recognize and destroy malignant, EGFRvIII-expressing glioma. In mice, we showed that EGFRvIII:CD3 bi-scFv effectively treats orthotopic patient-derived malignant glioma and syngeneic glioblastoma. Here, we developed a targeted assay for pharmacokinetic (PK) analysis of EGFRvIII:CD3 bi-scFv, a necessary step in the drug development process. Using microflow liquid chromatography coupled to a high resolution parallel reaction monitoring mass spectrometry, and data analysis in Skyline, we developed a bottom-up proteomic assay for quantification of EGFRvIII:CD3 bi-scFv in both plasma and whole blood. Importantly, a protein calibrator, along with stable isotope-labeled EGFRvIII:CD3 bi-scFv protein, were used for absolute quantification. A PK analysis in a CD3 humanized mouse revealed that EGFRvIII:CD3 bi-scFv in plasma and whole blood has an initial half-life of ∼8 min and a terminal half-life of ∼2.5 h. Our results establish a sensitive, high-throughput assay for direct quantification of EGFRvIII:CD3 bi-scFv without the need for immunoaffinity enrichment. Moreover, these pharmacokinetic parameters will guide drug optimization and dosing regimens in future IND-enabling and phase I studies of EGFRvIII:CD3 bi-scFv.

Abstract 166: Effects of Dietary Unsaturated Fat and Carbohydrate on the HDL Proteome and Metabolism of 9 HDL Proteins Across 6 HDL Size Fractions in Humans

Introduction: When dietary carbohydrate replaces fat, HDL-C and apoA1 decrease. The proportion in plasma of large HDL2 decreases while small HDL3 increases. These findings suggest that diet affects metabolically important attributes of HDL that produce the size changes. We studied in humans the effect of dietary carbohydrate and unsaturated fat on the HDL proteome and metabolism of 9 HDL proteins across 6 HDL sizes. Methods and Results: Twelve participants were placed on a controlled high unsaturated fat (HF) or high carbohydrate (HC) diet in a randomized crossover design. At the end of each 4-week diet period, subjects were infused with D3-Leu tracer, and blood was collected for 70 hrs. ApoA1-HDL was prepared by immunoaffinity purification, separated into 6 sizes α0, α1, α2, α3, preβ, and &lt;preβ by ND-PAGE, and in-gel trypsinized for mass spectrometry. We used label free quantification to characterize the HDL proteome. The proteome composition and distribution across the 6 HDL sizes were remarkably conserved in all subjects on both diets. Diet altered the abundance of several proteins on the major HDL sizes α2 and α3. The HC diet increased proteins involved in lipid metabolism (apoC3, apoC1, apoC2-C4) and acute phase response (SAA1, 4), and decreased antioxidant (PON1, 3) and protease inhibitor (SERPINA3, G1) proteins. We also monitored the metabolism of 9 proteins that likely affect HDL metabolism – apoA1, apoA2, apoA4, apoC3, apoE, apoM, apoJ, apoL1, and LCAT. We used high resolution parallel reaction monitoring and XPI software to measure tracer enrichment in these 9 proteins across the 6 HDL sizes. We found that the HC diet increased apoA2 and decreased apoA1 and apoA4 pool sizes on large HDL. The HC diet increased apoA1 and apoE turnover on large α1 and α2 and 3, respectively. Conclusions: Dietary carbohydrate when it replaces unsaturated fat alters the HDL proteome and metabolism of several HDL proteins on specific HDL sizes. Carbohydrate increases the abundance of proteins involved in lipid metabolism and the acute phase response, decreases antioxidant and protease inhibitor proteins, and enhances turnover of apoE and apoA1. This study suggests that diet modulates HDL function by affecting HDL composition and the metabolism of major HDL proteins.

Correction to Integrating MS1 and MS2 Scans in High-Resolution Parallel Reaction Monitoring Assays for Targeted Metabolite Quantification and Dynamic 13C-Labeling Metabolism Analysis.

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionNEXTORIGINAL ARTICLEThis notice is a correctionCorrection to Integrating MS1 and MS2 Scans in High-Resolution Parallel Reaction Monitoring Assays for Targeted Metabolite Quantification and Dynamic 13C-Labeling Metabolism AnalysisZhucui LiZhucui LiMore by Zhucui Li, Yujin LiYujin LiMore by Yujin Li, Wujiu ChenWujiu ChenMore by Wujiu Chen, Qichen CaoQichen CaoMore by Qichen Cao, Yufeng GuoYufeng GuoMore by Yufeng Guo, Ni WanNi WanMore by Ni Wan, Xiaolong JiangXiaolong JiangMore by Xiaolong Jiang, Yinjie J. TangYinjie J. TangMore by Yinjie J. Tang, Qinhong WangQinhong WangMore by Qinhong Wanghttp://orcid.org/0000-0003-0004-2721, and Wenqing Shui*Wenqing ShuiMore by Wenqing Shuihttp://orcid.org/0000-0002-5245-2477Cite this: Anal. Chem. 2018, 90, 8, 5509Publication Date (Web):March 28, 2018Publication History Published online28 March 2018Published inissue 17 April 2018https://doi.org/10.1021/acs.analchem.8b01316Copyright © 2018 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views1150Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (206 KB) Get e-Alerts Get e-Alerts

Polymeric ion exchange material based dispersive micro solid-phase extraction of lipophilic marine toxins in seawater followed by the Q Exactive mass spectrometer analysis using a scheduled high resolution parallel reaction monitoring

In recent years, harmful algal blooms have been occurring frequently all over the world, which caused a great negative impact on marine ecosystem, aquaculture and human health. Lipophilic marine toxins (LMTs) produced by certain harmful alga are closely related to diarrheic shellfish poisoning. In this study, a new sample preparation method was established by performing dispersive micro solid-phase extraction (DMSPE) procedure using mixed polymeric ion exchange material as the adsorbent for the simultaneous extraction of LMTs in seawater. To obtain the optimal extraction efficiency of LMTs, the key factors including the type and the amount of adsorbent, the type and volume of eluent, and the concentration of sodium chloride were investigated in detail. A good enrichment effect and weak matrix effects were observed after the DMSPE procedure. Compared with conventional solid-phase extraction sample preparation, the advantages of DMSPE procedure were present in convenience and low cost. In high resolution mass spectrometry (HRMS) detection, a new scheduled high resolution parallel reaction monitoring (PRM) mode was selected for qualitative and quantitative analysis to improve the selectivity and sensitivity of LMTs in seawater. Based on an effective combination of DMSPE procedure in sample preparation and high resolution PRM in data acquisition, a sufficiently low detection level (pg/L level) was achieved for the routine monitoring of LMTs in seawater.

Comprehensive Analysis of Acylcarnitine Species in db/db Mouse Using a Novel Method of High-Resolution Parallel Reaction Monitoring Reveals Widespread Metabolic Dysfunction Induced by Diabetes.

Acylcarnitines are exerting a variety of biological functions depending on the differences in lengths, saturation levels, and conjugation groups, which to a great extent contribute to the challenges of acylcarnitines quantifications due to various kinds of isomers. Here, we describe a novel method by using high-resolution parallel reaction monitoring (PRM) liquid chromatography-tandem mass spectrometry (LC-MS/MS). Both reversed-phase and normal-phase column were used in order to get accurate, reliable, widespread quantification of acylcarnitines, and without tedious sample preparation procedure. The method provided the most comprehensive acylcarnitine profile with high-resolution MS and MS/MS confirmation to date. A total of 117 acylcarnitines were detected from plasma and urine samples. The application of targeted profiling of acylcarnitines in db/m+ control and db/db diabetic mice indicated incomplete amino acid and fatty acid oxidation on diabetic mice. Interestingly, the reduction of medium odd-numbered chain acylcarnitines in urine samples was first observed between db/m+ and db/db mice. The high-resolution PRM method makes it possible to monitor the widespread metabolic changes of the acylcarnitines in response to stimuli. Besides, the accurate MS and MS/MS spectra data of the 117 acylcarnitines could be used as mass spectrometric resources for the identification of acylcarnitines.

Application of selected reaction monitoring and parallel reaction monitoring for investigation of HL-60 cell line differentiation.

Targeted mass spectrometry represents a powerful tool for investigation of biological processes. The convenient approach of selected reaction monitoring using stable isotope-labeled peptide standard (SIS) is widely applied for protein quantification. Along with this method, high-resolution parallel reaction monitoring has been increasingly used for protein targeted analysis. Here we applied two targeted approaches (selected reaction monitoring with SIS and label-free parallel reaction monitoring) to investigate expression of 11 proteins during all-trans retinoic acid-induced differentiation of HL-60 cells. In our experiments, we have determined the proteins expression ratio at 3, 24, 48, and 96 h after all-trans retinoic acid treatment in comparison with 0 h, respectively. Expression profiles of four proteins (VAV1, PRAM1, LYN, and CEBPB) were highly correlated ( r > 0.75) and FGR expression was detected on proteome level starting from 24 h by both techniques. For prominent differences (fold change ≥ 2) label-free parallel reaction monitoring is not inferior to selected reaction monitoring with isotopically labeled peptide standards. Differentially expressed proteins, that have been determined in our study, can be considered as potential drug targets for acute myeloid leukemia (AML) treatment.

Integrating MS1 and MS2 Scans in High-Resolution Parallel Reaction Monitoring Assays for Targeted Metabolite Quantification and Dynamic 13C-Labeling Metabolism Analysis.

Quantification of targeted metabolites, especially trace metabolites and structural isomers, in complex biological materials is an ongoing challenge for metabolomics. Initially developed for proteomic analysis, the parallel reaction monitoring (PRM) technique exploiting high-resolution MS2 fragment ion data has shown high promise for targeted metabolite quantification. Notably, MS1 ion intensity data acquired independently as part of each PRM scan cycle are often underutilized in the PRM assay. In this study, we developed an MS1/MS2-combined PRM workflow for quantification of central carbon metabolism intermediates, amino acids and shikimate pathway-related metabolites on an orthogonal QqTOF system. Concentration curve assessment revealed that exploiting both MS1 and MS2 scans in PRM analysis afforded higher sensitivity, wider dynamic range and better reproducibility than relying on either scan mode for quantification. Furthermore, Skyline was incorporated into our workflow to process the MS1/MS2 ion intensity data, and eliminate noisy signals and transitions with interferences. This integrated MS1/MS2 PRM approach was applied to targeted metabolite quantification in engineered E. coli strains for understanding of metabolic pathway modulation. In addition, this new approach, when first implemented in a dynamic 13C-labeling experiment, showed its unique advantage in capturing and correcting isotopomer labeling curves to facilitate nonstationary 13C-labeling metabolism analysis.

Abstract 538: Metabolism of Multiple Apolipoproteins Across HDL Size in Humans

Introduction: Studying the kinetics of several HDL apolipoproteins together may help elucidate their function in HDL metabolism. We present multicompartmental analysis describing the metabolism of several apolipoproteins across 5 HDL size fractions. Methods and Results: Three participants were infused with a bolus of D3-Leu tracer, and blood was collected for 70 hrs. ApoA-I-HDL was prepared by immunoaffinity purification, separated into 5 size fractions, preβ, α3, α2, α1, and α0, by ND-PAGE, and in-gel trypsinized for mass spectrometry. We monitored 7 proteins that likely affect HDL metabolism - apoA-I, apoA-II, apoE, apoM, apoC-III, apoA-IV, and apoD. We used stable isotope labeled peptide standards to quantify pool size and high resolution parallel reaction monitoring to measure tracer enrichment in these 7 proteins in the 5 HDL size fractions. These data were then used for multicompartmental analysis to describe the kinetic behaviors across HDL size for each apolipoprotein, with the exception of apoD. All size fractions with discernible enrichment curves for at least 2 participants were included in the final model for each apolipoprotein (Fig.1). The majority of apoA-I α HDL originated form the source compartment, presumably the liver and/or small intestine, while apoA-I preβ HDL came primarily from α3. Size expansion pathways (preβ to α1/2, and α3 to α2) contributed only slightly to apoA-I metabolism. Similarly to apoA-I on the α sizes, the majority of the other apolipoproteins appear on each HDL size fraction directly from the source compartment. Only minor flux pathways from smaller to larger HDL sizes were identified: apoA-II, α3 to α2; apoE, α3 to α2, and α3 to α1. Conclusions: This study supports the new model of HDL metabolism in which HDL and its attached apolipoproteins are metabolized mainly within each HDL size fraction. Pathways between HDL sizes only provide a small contribution to the metabolism of each apolipoprotein.

Targeting LAMP2 in human cerebrospinal fluid with a combination of immunopurification and high resolution parallel reaction monitoring mass spectrometry.

Background Alzheimer’s disease is the most common form of dementia. An increasing body of evidence suggests that endo-lysosomal dysfunction is a pathogenic mechanism of Alzheimer’s disease. Thus there is a potential for proteins involved in the normal function of endo-lysosomal vesicles to act as biomarkers of disease. Herein we focused on the lysosomal protein LAMP2 that is involved in chaperone mediated autophagy.ResultsUsing a combination of immunoprecipitation, digestion and nano-liquid chromatography tandem mass spectrometry we targeted and identified six tryptic LAMP2 peptides in human cerebrospinal fluid. Employing the identified proteotypic tryptic peptides a hybrid immunoprecipitation high resolution parallel reaction monitoring mass spectrometric method was developed for the relative quantitation of LAMP2. The method was evaluated in a number of experiments which defined the overall methodological as well as the analytical micro-liquid chromatography mass spectrometric intra- and inter-day variability. We identified an overall methodological peptide dependent intra-day variability of 8–16 %. The inter-day experiments showed similar results. The analytical contribution to the variation was minor with a coefficient of variation of 0.5–2.1 %, depending on the peptide. Using the developed method, with defined and limited variability, we report increased cerebrospinal fluid levels of three LAMP2 peptides in Alzheimer’s disease subjects (n = 14), as compared to non-Alzheimer’s disease controls (n = 14).ConclusionAltered LAMP2 levels in cerebrospinal fluid may indicate endo-lysosomal dysfunction in Alzheimer’s disease. However, further studies in larger cohorts comprised of well-defined patient materials are required. We here present a tool which can be used for exploring the relevance of the level of LAMP2 as a potential measure of lysosomal dysfunction in Alzheimer’s disease or other neurodegenerative diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-016-9104-2) contains supplementary material, which is available to authorized users.

Multiplexed, Scheduled, High-Resolution Parallel Reaction Monitoring on a Full Scan QqTOF Instrument with Integrated Data-Dependent and Targeted Mass Spectrometric Workflows.

Recent advances in commercial mass spectrometers with higher resolving power and faster scanning capabilities have expanded their functionality beyond traditional data-dependent acquisition (DDA) to targeted proteomics with higher precision and multiplexing. Using an orthogonal quadrupole time-of flight (QqTOF) LC-MS system, we investigated the feasibility of implementing large-scale targeted quantitative assays using scheduled, high resolution multiple reaction monitoring (sMRM-HR), also referred to as parallel reaction monitoring (sPRM). We assessed the selectivity and reproducibility of PRM, also referred to as parallel reaction monitoring, by measuring standard peptide concentration curves and system suitability assays. By evaluating up to 500 peptides in a single assay, the robustness and accuracy of PRM assays were compared to traditional SRM workflows on triple quadrupole instruments. The high resolution and high mass accuracy of the full scan MS/MS spectra resulted in sufficient selectivity to monitor 6-10 MS/MS fragment ions per target precursor, providing flexibility in postacquisition assay refinement and optimization. The general applicability of the sPRM workflow was assessed in complex biological samples by first targeting 532 peptide precursor ions in a yeast lysate, and then 466 peptide precursors from a previously generated candidate list of differentially expressed proteins in whole cell lysates from E. coli. Lastly, we found that sPRM assays could be rapidly and efficiently developed in Skyline from DDA libraries when acquired on the same QqTOF platform, greatly facilitating their successful implementation. These results establish a robust sPRM workflow on a QqTOF platform to rapidly transition from discovery analysis to highly multiplexed, targeted peptide quantitation.

High resolution parallel reaction monitoring with electron transfer dissociation for middle-down proteomics.

In recent years, middle-down proteomics has emerged as a popular technique for the characterization and quantification of proteins not readily amenable to typical bottom-up approaches. So far, all high resolution middle-down approaches are done in data-dependent acquisition mode, using both collision-induced dissociation or electron capture/transfer dissociation techniques. Here, we explore middle-down proteomics with electron transfer dissociation using a targeted acquisition mode, parallel reaction monitoring (PRM), on an Orbitrap Fusion. As an example of a highly modified protein, we used histone H3 fractions from untreated and DMSO-treated Murine ErythroLeukemia (MEL) cells. We first determined optimized instrument parameters to obtain high sequence coverage using a synthetic standard peptide. We then setup a combined method of both MS1 scans and PRM scans of the 20 most abundant combinations of methylation and acetylation of the +10 charge state of the N-terminal tail of H3. Weak cation exchange hydrophilic interaction chromatography was used to separate the N-terminal H3 tail, primarily, by its acetylation and, to a secondary degree, by its methylation status, which aided in the interpretation of the results. After deconvolution of the highly charged ions, peaks were annotated to a minimum set of 254 H3 proteoforms in the untreated and treated samples. Upon DMSO treatment, global quantitation changes from the MS1 level show a relative decrease of 2, 3, 4, and 5 acetylations and an increase of 0 and 1 acetylations. A fragment ion map was developed to visualize specific differences between treated and untreated samples. Taken together, the data presented here show that middle-down proteomics with electron transfer dissociation using PRM is a novel, attractive method for the effective analysis and quantification of large and highly modified peptides.

Abstract 322: HDL Dynamics in Circulation: Complexity of Protein Distribution and Metabolism Across HDL Size

Introduction: The composition of specific apolipoproteins may determine HDL functions. We present novel mass spectrometry (MS)-based methods that capture the absolute quantities and kinetics of 7 apolipoproteins in 5 HDL size fractions. Methods and Results: Three participants were recruited, infused with a bolus of D3-Leu tracer, and blood was collected for 70 hrs. ApoA-I-containing HDL was prepared by immunoaffinity purification, separated into 5 size fractions, preβ, α3, α2, α1, and α0 by ND-PAGE, and in-gel trypsinized for MS. We monitored 7 proteins that likely affect HDL metabolism - apoA-I, apoA-II, apoA-IV, apoC-III, apoD, apoE and apoM. Each protein pool size had a distinct distribution across the HDL sizes. ApoE and apoM were enriched in larger HDL, whereas apoC-III and apoA-IV were enriched in smaller HDL sizes. We evaluated the tracer enrichment curves of these 7 proteins in the 5 fractions using high resolution parallel reaction monitoring performed on a quadrupole Orbitrap (Thermo). The enrichment curves for each protein varied from each other by slope and time of peak enrichment (Fig. 1a). In contrast, the enrichment curves across HDL sizes for a single protein showed smaller, but likely meaningful, differences in either slope or time of peak enrichment. Irrespective of the HDL size on which it resides, apoE had the fastest FCR, followed by apoA-IV, and apoC-III. ApoA-I/A-II, apoM, and apoD had slower but similar FCRs (Fig. 1b). Conclusions: This study showed distinct distribution and kinetic behaviors of 7 HDL proteins across 5 HDL size fractions that were conserved in the three participants. These findings may help elucidate the functional role of these proteins and the HDL particles that contain them.