A Simplified Proteomics LC-MRM-MS Assay for Determination of apoE Genotypes in Plasma Samples.

Apolipoprotein E (apoE), a polymorphic plasma protein, plays a pivotal role in lipid transportation. The human apoE gene possesses three major alleles (ε2, ε3, and ε4), which differ by single amino acid (cysteine to arginine) substitutions. The ε4 allele represents the primary genetic risk factor for Alzheimer's disease (AD), whereas the ε2 allele protects against the disease. Knowledge of a patient's apoE genotype has high diagnostic value. A recent study has introduced an LC-MRM-MS-based proteomic approach for apoE isoform genotyping using stable isotope-labeled peptide internal standards (SIS). Here, our goal was to develop a simplified LC-MRM-MS assay for identifying apoE genotypes in plasma samples, eliminating the need for the use of SIS peptides. To determine the apoE genotypes, we monitored the chromatographic peak area ratios of isoform-specific peptides relative to a peptide that is common to all apoE isoforms. The assay results correlated well with the standard TaqMan allelic discrimination assay, and we observed a concordance between the two methods for all but three out of 172 samples. DNA sequencing of these three samples has confirmed that the results of the LC-MRM-MS method were correct. Thus, our simplified UPLC-MRM-MS assay is a feasible and reliable method for identifying apoE genotypes without using SIS internal standard peptides. The approach can be seamlessly incorporated into existing quantitative proteomics assays and kits, providing additional valuable apoE genotype information. The principle of using signal ratios of the protein isoform-specific peptides to the peptide common for all of the protein isoforms has the potential to be used for protein isoform determination in general.

Quantification of Caffeoylquinic Acids and Triterpenes as Targeted Bioactive Compounds of Centella asiatica in Extracts and Formulations by Liquid Chromatography Mass Spectrometry.

Centella asiatica (CA) is a culinary vegetable and well-known functional food that is widely used as a medicinal herb and dietary supplement. CA is rich in pentacyclic triterpenes (TTs), including asiaticoside (AS), madecassoside (MS) and the related aglycones asiatic acid (AA), madecassic acid (MA). Traditionally, TTs have been associated with the bioactivity and health promoting effect of CA. Recently, mono-caffeoylquinic acids (MonoCQAs) and di-caffeoylquinic acids (DiCQAs) have been found to contribute to the bioactivity of CA as well. This work reports an analytical strategy based on liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM-MS) for the simultaneous rapid and accurate quantification of 12 bioactive compounds in CA, namely AS, MS, AA, MA, 5-CQA, 4-CQA, 3-CQA, 1,3-DiCQA, 3,4-DiCQA, 1,5-DiCQA, 3,5-DiCQA, 4,5-DiCQA. Method selectivity, accuracy, precision, repeatability, robustness, linearity range, limit of detection (LOD), and limit of quantitation (LOQ) were validated. The validated LC-MRM-MS method has been successfully applied to quantify the 12 bioactive compounds in CA aqueous extracts and two related formulations: a standardized CA product (CAP) used in a phase I clinical trial and formulated CA rodent diets used in preclinical studies. The validated method allows us to support the standardization of CA products used for clinical trials and conduct routine LC-MRM-MS analyses of formulated preclinical diets to confirm correct levels of CA phytochemical markers.

An enhanced LC-MRM-MS platform for sensitive and simultaneous quantification of cyclic imines in shellfish.

Cyclic imines (CIs) produced by microalgae species and accumulating in the food chain of marine organisms are novel biotoxins that do not belong to the classical group of marine biotoxins. In the past, CIs were found only in limited areas, but in recent years, rapid changes in marine ecosystems have led to widespread CIs, increasing exposure to toxic risks. Monitoring of CIs is therefore required, but still analytically challenging due to the presence of high levels of analogues and interference from other lipophilic substances. Herein, we developed the LC/MRM-MS-based quantitative platform that can selectively enrich for marine-derived CIs and monitor seven CIs simultaneously: pinnatoxin (PnTX E, PnTX F, PnTX G), gymnodimine (GYM A), and spirolide (13-desMe SPX C, 13,19-didesMe SPX C, 20-Me SPX G). In particular, the combination of chromatographic separation by the hydrophobic nature of intrinsic residues of CIs with monitoring of CI structure-specific product ions generated by CID-MS/MS significantly improves the selectivity and sensitivity for quantitative analysis. Indeed, three CIs corresponding to PnTX G, GYM A, and 13-desMe SPX C could be successfully determined at the level of part-per-trillion (ppt) in three species of shellfish collected around the Korean Peninsula. Our analysis revealed that the expression of CIs in the Korean Peninsula was more influenced by the season rather than the species. This analytical platform with high sensitivity can be applied not only to marine biology but also to various other fields requiring CI analysis. Key Contribution: A highly sensitive analytical method for the simultaneous quantitation of cyclic imines based on LC/MRM-MS has been developed.

Amino acid analysis for peptide quantitation using reversed-phase liquid chromatography combined with multiple reaction monitoring mass spectrometry.

Amino acid analysis (AAA) can be used for absolute quantitation of standard peptides after acid hydrolysis using 6 M HCl. Obtained individual amino acids can then be quantified by liquid chromatography-mass spectrometry (LC-MS). Achieving baseline separation of non-derivatized amino acids is challenging when reversed-phase (RP) chromatography is used. Several derivatization methods are commonly utilized to address this issue; however, derivatization has several drawbacks, such as derivative instability and lack of reproducibility. Currently, separation of non-derivatized amino acids is typically done using HILIC, but HILIC has problems of poor reproducibility and long column equilibration times. We developed a method to quantify non-derivatized amino acids, including methionine and cysteine, from peptide hydrolysates by RP-LC-MS without special pre-treatment of the samples. Samples were spiked with certified isotopically labeled (13C- and/or 15N-) amino acids as internal standards. The amino acids released from acid hydrolysis were then analyzed by RP-UPLC-MRM-MS and quantified using the analyte/internal standard chromatographic peak area ratios. Peptide quantitation was based on the sum of the individual amino acid concentrations from the known peptide sequences. The resulting method did not require derivatization, used standard C18-based reversed-phase liquid chromatography, did not require external calibration, was robust, and was able to quantify all 17 amino acids for which we had internal standards, including the sulfur-containing amino acids, cysteine and methionine, in their respective oxidized forms. This simple and robust method enabled the absolute quantitation of standard peptides using only acid hydrolysis and a standard RP-UPLC-MRM-MS setup.

Conventional solid-state fermentation impacts the white lupin proteome reducing the abundance of allergenic peptides.

The demand for high-quality and sustainable protein sources is on the rise. Lupin is an emerging plant-based source of protein with health-enhancing properties; however, the allergenic potential of lupins limits their widespread adoption in food products. A combination of discovery and targeted quantitative proteome measurements was used to investigate the impact of solid-state fermentation induced by Rhizopus oligosporus on the proteome composition and allergenic protein abundances of white lupin seed. In total, 1,241 proteins were uniquely identified in the fermented sample. Moreover, the effectiveness of the solid-state fermentation in reducing the abundance of the tryptic peptides derived from white lupin allergens was demonstrated. Comparably, a greater decrease was noted for the major white lupin allergen based on ß-conglutin peptide abundances. Hence, conventional solid-state fermentation processing can be beneficial for reducing the potential allergenicity of lupin-based foods. This finding will open new avenues for unlocking the potential of this under-utilised legume.

Safe food through better labelling; a robust method for the rapid determination of caprine and bovine milk allergens.

Accidental milk cross-contamination is one of the most common causes for costly food recalls. Yet, quantifying trace-levels of allergen is time-consuming and current methods are not adapted for routine analyses making quality control for trace-level allergen content impractical. This perpetuates voluntary "may-contain" statements that are unhelpful for people suffering from food allergies. Here, we developed a rapid LC-MS method enabling milk allergen quantification by comparing all tryptic-peptides of major milk allergens. The bovine-specific αS-2 casein peptide and allergen-epitope NAVPITPTLNR provided excellent performance in sensitivity (LOD 1 mg.kg-1; LOQ 2 mg.kg-1) across various dairy products, good recovery rates in baked croissants (77% with a 10% inter-day RSD) and a linear range of 2-2,000 mg.kg-1. The method can be used for routine determination of trace-contamination with bovine milk allergen and the adulteration of high-value caprine dairy products with lower-value bovine milk products, protecting consumer trust and the growing population suffering from food allergies.

Comparison of protein extraction protocols and allergen mapping from black soldier fly Hermetia illucens.

Exploration of important insect proteins - including allergens - and proteomes can be limited by protein extraction buffer selection and the complexity of the proteome. Herein, LC-MS/MS-based proteomics experiments were used to assess the protein extraction efficiencies for a suite of extraction buffers and the effect of ingredient processing on proteome and allergen detection. Discovery proteomics revealed that SDS-based buffer yields the maximum number of protein groups from three types of BSF samples. Bioinformatic analysis revealed that buffer composition and ingredient processing could influence allergen detection. Upon applying multi-level filtering criteria, 33 putative allergens were detected by comparing the detected BSF proteins to sequences from public allergen protein databases. A targeted LC-MRM-MS assay was developed for the pan-allergen tropomyosin and used to assess the influence of buffer composition and ingredient processing using peptide abundance measurements. SIGNIFICANCE: We demonstrated that the selection of protein extraction buffer and the processing method could influence protein yield and cross-reactive allergen detection from processed and un-processed black soldier fly (BSF) samples. In total, 33 putative allergens were detected by comparing the detected BSF proteins to sequences from public allergen protein databases. An LC-MRM-MS assay was developed for tropomyosin, indicating the importance of buffer selection and processing conditions to reduce BSF samples' allergenicity.

Evaluation of the Major Seed Storage Proteins, the Conglutins, Across Genetically Diverse Narrow-Leafed Lupin Varieties.

Lupin seeds have an excellent nutritional profile, including a high proportion of protein and dietary fiber. These qualities make lupin seeds an ideal candidate to help meet the growing global demand for complementary sources of protein. Of consequence to this application, there are nutritional and antinutritional properties assigned to the major lupin seed storage proteins-referred to as α-, ß-, δ- and γ-conglutins The variation in the abundance of these protein families can impact the nutritional and bioactive properties of different lupin varieties. Hence, exploring the conglutin protein profiles across a diverse range of lupin varieties will yield knowledge that can facilitate the selection of superior genotypes for food applications or lupin crop improvement. To support this knowledge generation, discovery proteomics was applied for the identification of the 16 known conglutin subfamilies from 46 domestic and wild narrow-leafed lupin (NLL) genotypes. Consequently, the diversity of abundance of these proteins was evaluated using liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS). This comparative study revealed a larger variability for the ß- and δ-conglutin content across the lines under study. The absence/lower abundance of the ß2- to ß6-conglutin subfamilies in a subset of the domesticated cultivars led to substantially lower overall levels of the allergenic ß-conglutin content in these NLLs, for which the elevation of the other conglutin families were observed. The diversity of the conglutin profiles revealed through this study-and the identification of potential hypoallergenic genotypes-will have great significance for lupin allergic consumers, food manufactures as well as grain breeders through the future development of lupin varieties with higher levels of desirable bioactive proteins and lower allergen content.

TRACES: A Lightweight Browser for Liquid Chromatography-Multiple Reaction Monitoring-Mass Spectrometry Chromatograms.

In targeted metabolomic analysis using liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS), hundreds of MRMs are performed in a single run, yielding a large dataset containing thousands of chromatographic peaks. Automation tools for processing large MRM datasets have been reported, but a visual review of chromatograms is still critical, as real samples with biological matrices often cause complex chromatographic patterns owing to non-specific, insufficiently separated, isomeric, and isotopic components. Herein, we report the development of new software, TRACES, a lightweight chromatogram browser for MRM-based targeted LC-MS analysis. TRACES provides rapid access to all MRM chromatograms in a dataset, allowing users to start ad hoc data browsing without preparations such as loading compound libraries. As a special function of the software, we implemented a chromatogram-level deisotoping function that facilitates the identification of regions potentially affected by isotopic signals. Using MRM libraries containing precursor and product formulae, the algorithm reveals all possible isotopic interferences in the dataset and generates deisotoped chromatograms. To validate the deisotoping function in real applications, we analyzed mouse tissue phospholipids in which isotopic interference by molecules with different fatty-acyl unsaturation levels is known. TRACES successfully removed isotopic signals within the MRM chromatograms, helping users avoid inappropriate regions for integration.

Shotgun lipidomics combined targeted MRM reveals sphingolipid signatures of coronary artery disease.

Sphingolipids (SPLs) are bioactive lipids that manifest structural diversity and complexity in eukaryotes. However, the distributions and functions of these molecules in mammalian tissues/cells have not been systematically investigated. Herein, we integrated shotgun lipidomics with targeted LC-MRM/MS approach to comprehensively analyze SPL species in various biological samples with high accuracy. Preliminarily, 1311 SPL molecules were identified in 18 kinds of mammalian samples, including 3 groups of human sera, 10 mouse tissues and 5 cell lines via 26 sphingoid long-chain bases scanning. The sphingolipidome compositions and distributions were systematically characterized and distinct qualitative and quantitative profiles were clearly exhibited in various samples, indicating unique biological functions of the sphingolipidomes. Next, targeted SPLs analysis by LC-MRM/MS with critical criteria monitoring two characteristic fragments of one precursor was applied to human serum samples from 24 coronary artery disease (CAD) patients and 12 healthy controls, which successfully quantified 170 SPL molecules. Ten novel SPL molecules were discovered as a potential diagnostic panel for CAD patients via multivariate exploratory receiver operating characteristic curve-based biomarker analysis. The diagnostic panel with the 10 SPL molecules achieved 97.2% accuracy, with a favorable auxiliary diagnostic value (AUC = 1.000), for the detection of CAD. These results clearly support the sphingolipidomic approach in application to discovering disease biomarker panel as well as deep investigation of biological functions of complex SPLs in mammalian samples.

Development and Provisional Validation of a Multiplex LC-MRM-MS Test for Timely Kidney Injury Detection in Urine.

Kidney injury is a complication frequently encountered in hospitalized patients. Early detection of kidney injury prior to loss of renal function is an unmet clinical need that should be targeted by a protein-based biomarker panel. In this study, we aim to quantitate urinary kidney injury biomarkers at the picomolar to nanomolar level by liquid chromatography coupled to tandem mass spectrometry in multiple reaction monitoring mode (LC-MRM-MS). Proteins were immunocaptured from urinary samples, denatured, reduced, alkylated, and digested into peptides before LC-MRM-MS analysis. Stable-isotope-labeled peptides functioned as internal standards, and biomarker concentrations were attained by an external calibration strategy. The method was evaluated for selectivity, carryover, matrix effects, linearity, and imprecision. The LC-MRM-MS method enabled the quantitation of KIM-1, NGAL, TIMP2, IGFBP7, CXCL9, nephrin, and SLC22A2 and the detection of TGF-ß1, cubilin, and uromodulin. Two to three peptides were included per protein, and three transitions were monitored per peptide for analytical selectivity. The analytical carryover was <1%, and minimal urine matrix effects were observed by combining immunocapture and targeted LC-MRM-MS analysis. The average total CV of all quantifier peptides was 26%. The linear measurement range was determined per measurand and found to be 0.05-30 nmol/L. The targeted MS-based method enables the multiplex quantitation of low-abundance urinary kidney injury biomarkers for future clinical evaluation.

Comparison of Proteomic Technologies for Blood-Based Detection of Colorectal Cancer.

Blood-based protein biomarkers are increasingly being explored as supplementary or efficient alternatives for population-based screening of colorectal cancer (CRC). The objective of the current study was to compare the diagnostic potential of proteins measured with different proteomic technologies. The concentrations of protein biomarkers were measured using proximity extension assays (PEAs), liquid chromatography/multiple reaction monitoring-mass spectrometry (LC/MRM-MS) and quantibody microarrays (QMAs) in plasma samples of 56 CRC patients and 99 participants free of neoplasms. In another approach, proteins were measured in serum samples of 30 CRC cases and 30 participants free of neoplasm using immunome full-length functional protein arrays (IpAs). From all the measurements, 9, 6, 35 and 14 protein biomarkers overlapped for comparative evaluation of (a) PEA and LC/MRM-MS, (b) PEA and QMA, (c) PEA and IpA, and (d) LC/MRM-MS and IpA measurements, respectively. Correlation analysis was performed, along with calculation of the area under the curve (AUC) for assessing the diagnostic potential of each biomarker. DeLong's test was performed to assess the differences in AUC. Evaluation of the nine biomarkers measured with PEA and LC/MRM-MS displayed correlation coefficients >+0.6, similar AUCs and DeLong's p-values indicating no differences in AUCs for biomarkers like insulin-like growth factor binding protein 2 (IGFBP2), matrix metalloproteinase 9 (MMP9) and serum paraoxonase lactonase 3 (PON3). Comparing six proteins measured with PEA and QMA showed good correlation and similar diagnostic performance for only one protein, growth differentiation factor 15 (GDF15). The comparison of 35 proteins measured with IpA and PEA and 14 proteins analyzed with IpA and LC/MRM-MS revealed poor concordance and comparatively better AUCs when measured with PEA and LC/MRM-MS. The comparison of different proteomic technologies suggests the superior performance of novel technologies like PEA and LC/MRM-MS over the assessed array-based technologies in blood-protein-based early detection of CRC.

Protein extraction protocols for optimal proteome measurement and arginine kinase quantitation from cricket Acheta domesticus for food safety assessment.

Insects have been consumed by people for millennia and have recently been proposed as a complementary, sustainable source of protein to feed the world's growing population. Insects and crustaceans both belong to the arthropod family. Crustacean (shellfish) allergies are common and potentially severe; hence, the cross-reactivity of the immune system with insect proteins is a potential health concern. Herein, LC-MS/MS was used to explore the proteome of whole, roasted whole and roasted powdered cricket products. Eight protein extraction protocols were compared using the total number of protein and distinct peptide identifications. Within these data, 20 putative allergens were identified, of which three were arginine kinase (AK) proteoforms. Subsequently, a multiple reaction monitoring MS assay was developed for the AK proteoforms and applied to a subset of extracts. This targeted assay demonstrated that allergen abundance/detectability varies according to the extraction method as well as the food processing method.

A UPLC-MRM-MS method for comprehensive profiling of Amadori compound-modified phosphatidylethanolamines in human plasma.

Phosphatidylethanolamines (PEs) are targets of non-enzymatic glycation, a chemical process that occurs between glucose and primary amine-containing biomolecules. As the early-stage non-enzymatic glycation products of PE, Amadori-PEs are implicated in the pathogenesis of various diseases. However, only a few Amadori-PE molecular species have been identified so far; a comprehensive profiling of these glycated PE species is needed to establish their roles in disease pathology. Herein, based on our previous work using liquid chromatography-coupled neutral loss scanning and product ion scanning tandem mass spectrometry (LC-NLS-MS and LC-PIS-MS) in tandem, we extend identification of Amadori-PE to the low-abundance species, which is facilitated by using plasma lipids glycated in vitro. The confidence of identification is improved by high-resolution tandem mass spectrometry and chromatographic retention time regression. A LC-coupled multiple reaction monitoring mass spectrometry (LC-MRM-MS) assay is further developed for more sensitive quantitation of the Amadori compound-modified lipids. Using synthesized stable isotope-labeled Amadori lipids as internal standards, levels of 142 Amadori-PEs and 33 Amadori-LysoPEs are determined in the NIST human plasma standard reference material. These values may serve as an important reference for future investigations of Amadori-modified lipids in human diseases.

Chemerin isoform analysis in human biofluids using an LC/MRM-MS-based targeted proteomics approach with stable isotope-labeled standard.

Targeted proteomics has advantages over earlier conventional technologies for protein detection. We developed and validated an LC/MRM-MS-based targeted proteomic method combined with immunoaffinity precipitation for the enrichment and detection of low abundance chemerin isoforms in human biofluids. After tryptic digestion, each chemerin isoform was characterized by isoform-specific peptides, and the absolute quantification was achieved by using stable isotope-labeled peptides as internal standards. In serum, follicular fluid and synovial fluid, a total of 6 chemerin isoforms were identified and quantified, among which a novel natural isoform 153Q was discovered for the first time. The relative content of the six chemerin isoforms in human serum was 157S ≫ 156F ≫ 158K > 154F ≥ 155A > 153Q in the ratio of 25:17:5:2.5:2.2:1, respectively. The absolute contents were in the range of 88-3.5 ng/mL. This distribution remained consistent among the 3 biofluids analyzed. Total chemerin were found to be increased in both polycystic ovary syndrome (serum and follicular fluid) and rheumatoid arthritis (serum) patients. However, chemerin isoform analysis revealed that only 156F & 157S were increased in the former, while 155A, 156F & 157S were increased in the latter. This demonstrates the potential of this method in detailed characterization of changes in chemerin isoforms that may be of clinical relevance.

Comprehensive Characterization and Relative Quantification of α-Amylase/Trypsin Inhibitors from Wheat Cultivars by Targeted HPLC-MS/MS.

The α-amylase/trypsin inhibitors (ATIs) are discussed as being responsible for non-celiac wheat sensitivity (NCWS), besides being known as allergenic components for baker's asthma. Different approaches for characterization and quantification including proteomics-based methods for wheat ATIs have been documented. In these studies generally the major ATIs have been addressed. The challenge of current study was then to develop a more comprehensive workflow encompassing all reviewed wheat-ATI entries in UniProt database. To substantially test proof of concept, 46 German and Turkish wheat samples were used. Two extractions systems based on chloroform/methanol mixture (CM) and under buffered denaturing conditions were evaluated. Three aspects were optimized, tryptic digestion, chromatographic separation, and targeted tandem mass spectrometric analysis (HPLC-MS/MS). Preliminary characterization with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) documented the purity of the extracted ATIs with CM mixture and the amylase (60-80%)/trypsin (10-20%) inhibition demonstrated the bifunctional activity of ATIs. Thirteen (individual/common) biomarkers were established. Major ATIs (7-34%) were differently represented in samples. Finally, to our knowledge, the proposed HPLC-MS/MS method allowed for the first time so far the analysis of all 14 reviewed wheat ATI entries reported.

Assessment of Serological Early Biomarker Candidates for Lung Adenocarcinoma by using Multiple Reaction Monitoring-Mass Spectrometry.

PURPOSE: Plasma markers that enable diagnosis in the early stage of lung cancer is not discovered. A liquid chromatography multiple reaction monitoring-mass spectrometry (LC-MRM-MS) assay for identifying potential early marker proteins for lung adenocarcinoma is developed. EXPERIMENTAL DESIGN: LC-MRM-MS assay is used for measuring the level of 35 candidate peptides in plasma from 102 lung adenocarcinoma patients (including n = 50, 16, 24, and 12 in stage I, II, III, and IV, respectively.) and 84 healthy controls. Stable isotope labeled standard peptides are synthesized to accurately measure the amount of these proteins. RESULTS: Seven proteins are able to distinguish stage I patients from controls. These proteins are combined in to a protein marker panel which improve the sensitivity to discriminate stage I patients from controls with cross-validated area under the curve = 0.76. Besides, it is found that low expression of eukaryotic initiation factor 4A-I and high expression of lumican show significantly poor prognosis in overall survival (p = 0.012 and 0.0074, respectively), which may be used as prognostic biomarkers for lung cancer. CONCLUSIONS AND CLINICAL RELEVANCE: Proteins highlighted here may be used for early detection of lung adenocarcinoma or therapeutics development after validation in a larger cohort.

Simultaneous quantification of free fatty acids and acylcarnitines in plasma samples using dansylhydrazine labeling and liquid chromatography-triple quadrupole mass spectrometry.

Free fatty acid (FFA) and acylcarnitine (AcCar) are key elements of energy metabolism. Dysregulated levels of FFA and AcCar are associated with genetic defects and other metabolic disorders. Due to differences in the physicochemical properties of these two classes of compounds, it is challenging to quantify FFA and AcCar in human plasma using a single method. In this work, we developed a chemical isotope labeling (CIL)-based liquid chromatography-multiple reaction monitoring (LC-MRM) method to simultaneously quantify FFA and AcCar. Dansylhydrazine (DnsHz) was used to label the carboxylic acid moiety on FFA and AcCar. This resulted in the formation of a permanently charged ammonium ion for facile ionization in positive ionization mode and higher hydrophobicity for enhanced retention of short-chain analogs on reversed-phase LC columns and enabled absolute quantification by using heavy labeled DnsHz analogs as internal standards. Labeling conditions including the concentration and freshness of cross-linker, reaction time, and temperature were optimized. This method can successfully quantify all short-, medium- and long-chain FFAs and AcCars with greatly enhanced sensitivity. Using this method, 25 FFAs and 13 AcCars can be absolutely quantified and validated in human plasma samples within 12 min. Simultaneous quantification of FFA and AcCar enabled by this CIL-based LC-MRM method facilitates the investigation of fatty acid metabolism and has potential in clinical applications.

Evaluation and Validation of Plasma Proteins Using Two Different Protein Detection Methods for Early Detection of Colorectal Cancer.

OBJECTIVE: Plasma protein biomarkers could be an efficient alternative for population-based screening for early detection of colorectal cancer (CRC). The objective of this study was to evaluate and validate plasma proteins individually and as a signature for early detection of CRC. METHODS: In a three-stage design, proteins were measured firstly by liquid chromatography/multiple reaction monitoring-mass spectrometry (LC/MRM-MS) and later by proximity extension assay (PEA) in a discovery set consisting of 96 newly diagnosed CRC cases and 94 controls free of neoplasms at screening colonoscopy. Two algorithms (one for each measurement method) were derived by Lasso regression and .632+ bootstrap based on 11 proteins that were included in both the LC/MRM-MS and PEA measurements. Additionally, another algorithm was constructed from the same eleven biomarkers plus amphireglin, the most promising protein marker in the PEA measurements that had not been available from the LC/MRM-MS measurements. Lastly the three prediction signatures were validated with PEA in independent samples of participants of screening colonoscopy (CRC (n = 56), advanced adenoma (n = 101), and participants free of neoplasm (n = 102)). RESULTS: The same four proteins were included in all three prediction signatures; mannan binding lectin serine protease 1, osteopontin, serum paraoxonase lactonase 3 and transferrin receptor protein 1, and the third prediction signature additionally included amphiregulin. In the independent validation set from a true screening setting, the five-marker blood-based signature including AREG presented areas under the curves of 0.82 (95% CI, 0.74-0.89), 0.86 (95% CI, 0.77-0.92) and 0.76 (95% CI, 0.64-0.86) for all, early and late stages CRC, respectively. CONCLUSION: Two different measurement methods consistently identified four protein markers and an algorithm additionally including amphiregulin, a marker measured by PEA only, showed promising performance for detecting early stage CRC in an independent validation in a true screening setting. These proteins may be potential candidates for blood-based tests for early detection of CRC.

Comprehensive analysis of oxylipins in human plasma using reversed-phase liquid chromatography-triple quadrupole mass spectrometry with heatmap-assisted selection of transitions.

Oxylipins, a subclass of lipid mediators, are metabolites of various polyunsaturated fatty acids with crucial functions in regulation of systemic inflammation. Elucidation of their roles in pathological conditions requires accurate quantification of their levels in biological samples. We refined an ultra-performance liquid chromatography-multiple reaction monitoring-mass spectrometry (UPLC-MRM-MS)-based workflow for comprehensive and specific quantification of 131 endogenous oxylipins in human plasma, in which we optimized LC mobile phase additives, column, and gradient conditions. We employed heatmap-assisted strategy to identify unique transitions to improve the assay selectivity and optimized solid phase extraction procedures to achieve better analyte recovery. The method was validated according to FDA guidelines. Overall, 94.4% and 95.7% of analytes at tested concentrations were within acceptable accuracy (80-120%) and precision (CV < 15%), respectively. Good linearity for most analytes was obtained with R2 > 0.99. The method was also validated using a standard reference material-SRM 1950 frozen human plasma to demonstrate inter-lab compatibility. Graphical abstract ᅟ.