Multiplex Assay to Determine Acute Phase Proteins in Modified Live PRRSV Vaccinated Pigs.

Acute phase protein (APP) response to vaccine challenges is an attractive alternative to natural infection for identifying pigs with increased disease resilience and monitoring the productive performance. Currently, the methods used for APP quantification are diverse and often based on techniques that use antibodies that are not necessarily pig specific. The objective of this work is the development of a method based on a UPLC-SRM/MS system for simultaneous determination of haptoglobin, apolipoprotein A1, C-reactive protein, pig-major acute protein, and serum amyloid A and its application in pigs to monitor the effect of a vaccine administered against porcine reproductive and respiratory syndrome virus (PRRSV). With the aim of tracing the complete analytical process for each proteotypic peptide, a synthetic QconCat polypeptide construct was designed. It was possible to develop an SRM method including haptoglobin, apolipoprotein A1, pig-MAP, and serum amyloid A1. The PRRSV vaccine only affected haptoglobin. The pigs with positive viremia tended to show higher values than negative pigs, reaching significant differences in the three haptoglobin SRM-detected peptides but not with the data acquired by immunoenzymatic and spectrophotometric assays. These results open the door to the use of SRM to accurately monitor APP changes in experimental pigs.

A Novel Peptidome Technology for the Diagnosis of Mild Cognitive Impairment and Alzheimer's Disease by Selected Reaction Monitoring.

Background: With the aging of populations worldwide, Alzheimer's disease (AD) has become a concern due to its high prevalence and the continued lack of established treatments. Early diagnosis is required as a preventive intervention to modify the disease's progression. In our previous study, we performed peptidomic analysis of serum samples obtained from AD patients and age-matched healthy subjects to seek peptide biomarker candidates for AD by using BLOTCHIP-MS analysis, and identified four peptides as AD biomarker candidates. Objective: The objective was to validate the serum biomarker peptides to distinguish mild cognitive impairment (MCI) and AD in comparison to cognitively healthy controls using a new peptidome technology, the Dementia Risk Test. Methods: We enrolled 195 subjects with normal cognitive function (NC; n = 70), MCI (n = 55), and AD (n = 70), The concentrations of cognitive impairment marker peptides (Fibrinogen α chain (FAC), Fibrinogen ß chain (FBC), Plasma protease C1 inhibitor (PPC1I), α2-HS-glycoprotein (AHSG)) were quantified by using a selected reaction monitoring assay based on liquid chromatography-MS/MS. Results: The present study confirmed that three peptides, FAC, FBC, and PPC1I, were significantly upregulated during the onset of AD. This three-peptide set was both highly sensitive in determining AD (sensitivity: 85.7%, specificity: 95.7%, AUC: 0.900) and useful in distinguishing MCI (sensitivity: 61.8%, specificity: 98.6%, AUC: 0.824) from NC. Conclusions: In this validation study, we confirmed the high diagnostic potential of the three peptides identified in our previous study as candidate serum biomarkers for AD. The Dementia Risk Test may be a powerful tool for detecting AD-related pathological changes.

Applicability of selected reaction monitoring for precise screening tests.

INTRODUCTION: The proactive identification of diseases through screening tests has long been endorsed as a means to preempt symptomatic onset. However, such screening endeavors are fraught with complications, such as diagnostic inaccuracies, procedural risks, and patient unease during examinations. These challenges are amplified when screenings for multiple diseases are administered concurrently. Selected Reaction Monitoring (SRM) offers a unique advantage, allowing for the high-throughput quantification of hundreds of analytes with minimal interferences. AREAS COVERED: Our research posits that SRM-based assays, traditionally tailored for single-disease biomarker profiling, can be repurposed for multi-disease screening. This innovative approach has the potential to substantially alleviate time, labor, and cost demands on healthcare systems and patients alike. Nonetheless, there are formidable methodological hurdles to overcome. These include difficulties in detecting low-abundance proteins and the risk of model overfitting due to the multiple functionalities of single proteins across different disease spectrums - issues especially pertinent in blood-based assays where detection sensitivity is constrained. As we move forward, technological strides in sample preparation, online extraction, throughput, and automation are expected to ameliorate these limitations. EXPERT OPINION: The maturation of mass spectrometry's integration into clinical laboratories appears imminent, positioning it as an invaluable asset for delivering highly sensitive, reproducible, and precise diagnostic results.

Use of Nonhuman Sera as a Highly Cost-Effective Internal Standard for Quantitation of Multiple Human Proteins Using Species-Specific Tryptic Peptides: Applicability in Clinical LC-MS Analyses.

Quantitation of proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is complex, with a multiplicity of options ranging from label-free techniques to chemically and metabolically labeling proteins. Increasingly, for clinically relevant analyses, stable isotope-labeled (SIL) internal standards (ISs) represent the "gold standard" for quantitation due to their similar physiochemical properties to the analyte, wide availability, and ability to multiplex to several peptides. However, the purchase of SIL-ISs is a resource-intensive step in terms of cost and time, particularly for screening putative biomarker panels of hundreds of proteins. We demonstrate an alternative strategy utilizing nonhuman sera as the IS for quantitation of multiple human proteins. We demonstrate the effectiveness of this strategy using two high abundance clinically relevant analytes, vitamin D binding protein [Gc globulin] (DBP) and albumin (ALB). We extend this to three putative risk markers for cardiovascular disease: plasma protease C1 inhibitor (SERPING1), annexin A1 (ANXA1), and protein kinase, DNA-activated catalytic subunit (PRKDC). The results show highly specific, reproducible, and linear measurement of the proteins of interest with comparable precision and accuracy to the gold standard SIL-IS technique. This approach may not be applicable to every protein, but for many proteins it can offer a cost-effective solution to LC-MS/MS protein quantitation.

A method for measuring serum levels of melanin-associated indole metabolites using LC-MS/MS and its application to malignant melanoma.

BACKGROUND AND AIMS: With the development of novel therapies for advanced malignant melanoma (MM), biomarkers that can accurately reflect the progression of MM are needed. Serum levels of melanin-related indole metabolites such as 5-hydroxy-6-methoxyindole-2-carboxylic acid (5H6MI2C) and 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C) are potential biomarkers for MM. Here, we describe the development of a mass spectrometry (MS)-based assay to determine serum levels of 5H6MI2C and 6H5MI2C. MATERIALS AND METHODS: We developed a stable isotope dilution-selective reaction monitoring-MS protocol using liquid chromatography tandem mass spectrometry (LC-MS/MS) to measure human serum 5H6MI2C and 6H5MI2C levels. Analytical evaluations of the method were performed and the method was applied to serum samples from MM patients (n = 81). RESULTS: The method established in this study showed high reproducibility and linearity. This novel method also found that serum 6H5MI2C levels were significantly elevated in patients with metastatic MM compared to those with non-metastatic MM. Unfortunately, 5H6MI2C did not show a comparable significant difference. CONCLUSION: We successfully established measurement methods for serum 5H6MI2C and 6H5MI2C levels using LC-MS/MS. Serum 6H5MI2C levels offer a potential marker for MM.

Use of quantitative mass spectrometry-based proteomics and ELISA to compare the alpha 2 macroglobulin concentration in equine blood-based products processed by three different orthobiologic devices.

Introduction: Alpha 2 macroglobulin (A2M), a multi-functional protein in the plasma protease inhibitor class, regulates proinflammatory cytokines and the clearance of chondrodestructive enzymes in cases of joint injury and osteoarthritis (OA). The purpose of this study was to compare A2M concentrations in equine plasma samples processed by three commercial devices developed for stall-side regenerative joint therapy. Methods: Plasma samples were obtained from healthy adult horses (N = 13). Mass spectrometry analysis was used to determine the concentration of protein analytes in each sample. Selected reaction monitoring measured a specific A2M peptide as a surrogate of the whole A2M protein. A2M concentrations produced by each test device were compared for two sample types: a pre-concentrate or platelet-poor (PP) component and a final component for use in the horse. Results: There was no significant difference (p > 0.05) in the geometric mean (GM) concentration of A2M in the final concentration samples produced by the Alpha2EQ® device (N horses = 13) and the single-centrifugation PP samples produced by the Pro-Stride® APS (autologous protein solution) device (N = 13) and the Restigen® PRP (platelet-rich plasma) device (N = 11). When A2M content in final concentration samples produced by each device was compared, the Pro-Stride APS and Restigen PRP samples had significantly greater GM A2M content (p < 0.0001) compared to the Alpha2EQ samples, and the Pro-Stride APS final concentration samples had significantly greater GM A2M concentration (p < 0.0001) versus that for the Restigen PRP final samples. Discussion: This comparison demonstrated that the volume and A2M concentration of an Alpha2EQ final concentrate are no different than the volume and concentration of A2M in the PP from Pro-Stride or Restigen devices.

Quantitation of phenylalanine and tyrosine from dried Blood/Plasma spots with impregnated stable isotope internal standards (SIIS) by FIA-SRM.

BACKGROUND: Dried Blood Spot (DBS) analysis has been used for identification and quantification of diseases and disorders in large populations. Simply collecting blood or plasma samples on cotton paper, followed with an organic solvent extraction, many small molecules can be detected and quantified. In a typical procedure of DBS analysis in newborn screening, stable isotope internal standards (SIIS) are added to extraction solvent as a reference. However, this way of employing SIIS does not reflect extraction efficiency, or protein binding issues, nor does it reflect potential degradation that could occur. In addition, punched-out discs from larger DBS are known to have imprecision typically ≥ 15%. METHODS: We developed and tested an approach, internal quantitative DBS (iqDBS), which delivers an exact volume of whole blood or plasma to a paper disc that is impregnated with a dried concentration of SIIS for quantitation. Amino acids were derivatized to make butyl esters and measured using Flow Injection Analysis with Selected Reaction Monitoring (FIA-SRM). RESULTS: We demonstrated with phenylalanine and tyrosine improved sensitivity and accuracy by applying iqDBS. CONCLUSIONS: We established a new method for quantitative analysis of small molecules from dried blood spots that incorporates stable isotope internal standard at the time of blood collection.

Targeted proteomics data interpretation with DeepMRM.

Targeted proteomics is widely utilized in clinical proteomics; however, researchers often devote substantial time to manual data interpretation, which hinders the transferability, reproducibility, and scalability of this approach. We introduce DeepMRM, a software package based on deep learning algorithms for object detection developed to minimize manual intervention in targeted proteomics data analysis. DeepMRM was evaluated on internal and public datasets, demonstrating superior accuracy compared with the community standard tool Skyline. To promote widespread adoption, we have incorporated a stand-alone graphical user interface for DeepMRM and integrated its algorithm into the Skyline software package as an external tool.

High throughput LC/ESI-MS/MS method for simultaneous analysis of 20 oral molecular-targeted anticancer drugs and the active metabolite of sunitinib in human plasma.

Many types of oral molecular-targeted anticancer drugs are clinically used in cancer genomic medicine. Combinations of multiple molecular-targeted anticancer drugs are also being investigated, expecting to prolong the survival of patients with cancer. Therapeutic drug monitoring of oral molecular-targeted drugs is important to ensure efficacy and safety. A liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has been used for simultaneous determination of these drugs in human plasma. However, the sensitivity of mass spectrometers and differences in the therapeutic range of drugs have rendered the development of simultaneous LC/ESI-MS/MS methods difficult. In this study, a simultaneous quantitative method for 20 oral molecular-targeted anticancer drugs and the active metabolite of sunitinib was developed based on the results of linear range shifts of the calibration curves using four ion abundance adjustment techniques (collision energy defects, in-source collision-induced dissociation, secondary product ion selected reaction monitoring, and isotopologue selected reaction monitoring). The saturation of the detector for the seven analytes was resolved by incorporating optimal ion abundance adjustment techniques. Furthermore, the reproducibility of this method was confirmed in validation tests, and plasma from patients was measured by this method to demonstrate its usefulness in actual clinical practice. This analytical method is expected to make a substantial contribution to the promotion of personalized medicine in the future.

Targeted Mass Spectrometry Assays for Specific Quantification of Urinary proPSA Isoforms.

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.

Malondialdehyde-Induced Post-translational Modifications in Hemoglobin of Smokers by NanoLC-NSI/MS/MS Analysis.

Malondialdehyde (MDA) is the most abundant α,ß-unsaturated aldehyde generated from endogenous peroxidation of polyunsaturated fatty acids and is present in cigarette smoke. Post-translational modifications of blood hemoglobin can serve as biomarkers for exposure to chemicals. In this study, two types of MDA-induced modifications, the N-propenal and the dihydropyridine (DHP), were identified at multiple sites in human hemoglobin digest by the high-resolution mass spectrometry. The N-propenal and the DHP types of modification led to the increase of 54.0106 and 134.0368 amu, respectively, at the N-terminal and lysine residues. Among the 21 MDA-modified peptides, 14 with dose-response to MDA concentrations were simultaneously quantified in study subjects by the nanoflow liquid chromatography nanoelectrospray ionization tandem mass spectrometry under selected reaction monitoring (nanoLC-NSI-MS/MS-SRM) without prior enrichment. The results showed that the modifications of the N-propenal-type at α-Lys-11, α-Lys-16, α-Lys-61, ß-Lys-8, and ß-Lys-17, as well as the DHP-type at the α-N-terminal valine, are significantly higher in hemoglobin isolated from the blood of smokers than in nonsmoking individuals. This is the first report to identify and quantify multiple sites of MDA-induced modifications in human hemoglobin from peripheral blood. Our results suggest that the MDA-derived modifications on hemoglobin might represent valuable biomarkers for MDA-induced protein damage.

Quantification and identification analysis of Ziziphus jujuba Mill. peel pigmentation at different developmental stages.

The fruit peel of a color mutant jujube cultivar, 'Sanbianhong' (SBF), was investigated using an ultra-high performance liquid chromatography quadrupole Orbitrap mass spectrometry (UHPLC-Q-Orbitrap MS) at five ripening stages (S1, Young fruit stage; S2, swelling stage; S3, white-mature stage; S4, pre-mature stage and S5, mature stage). Lutein, ß-carotene, chlorophyll a, chlorophyll b, and 13 anthocyanins were identified. Chlorophyll a and cyanidin 3-O-galactoside were considered key color metabolites in S1 with the content of 1.083 mg/g of fresh weight (FW) and 4.585 mg/g of FW, respectively. Delphinidin (0.488 mg/g FW) and cyanidin (6.259 mg/g FW) were identified as the key pigments in S3. Delphinidin 3-O-glucoside (0.256 mg/g FW) was identified as the key anthocyanin in maturity S5. Herein, the identification and quantitation of pigment-related metabolites of SBF were studied for the first time, and the results provide a theoretical basis for understanding the pigment changes of jujube fruit during ripening.

Quantitative Detection of Protein Splice Variants by Selected Reaction Monitoring (SRM) Mass Spectrometry.

Molecular diversification of the cellular proteome through alternative splicing has emerged as an important biological principle. However, the lack of tools to specifically detect and quantify proteoforms (Smith et al., Nat Methods 10:186-187, 2013) is a major impediment to functional studies. Recently, biological mass spectrometry (MS) has undergone impressive advances (Mann, Nat Rev Mol Cell Biol 17:678, 2016), including the generation of a highly diverse set of biological applications (Aebersold and Mann, Nature 537:347-355, 2016), and has demonstrated to be an essential tool to address many biological questions (Savitski et al., Science 346:1255784, 2014; Rinner et al., Nat Methods 5:315-318, 2008). In particular, targeted LC-MS, with its high selectivity and specificity, is ideally suited for the precise and sensitive quantification of specific proteins and their proteoforms (Picotti and Aebersold, Nat Methods 9:555-566, 2012). We describe in detail the application of this workflow applied to dissect the molecular diversity of the synaptic adhesion proteins and their splicing-derived proteoforms (Schreiner et al., Elife 4:e07794, 2015).

A rapid LC-MS/MS assay for detection and monitoring of underivatized branched-chain amino acids in maple syrup urine disease.

Introduction: Quantitation of the isomeric branched-chain amino acids (BCAA; valine, alloisoleucine, isoleucine, leucine) is a challenging task that typically requires derivatization steps or long runtimes if a traditional chromatographic method involving a ninhydrin ion pairing reagent is used. Objectives: To develop and perform clinical validation of a rapid, LC-MS/MS-based targeted metabolomics assay for detection and monitoring of underivatized BCAA in human plasma. Methods: Various columns and modes of chromatography were tested. The final optimized method utilized mixed mode chromatography with an Intrada column under isocratic condition. Sample preparation utilized the 96-well format. Briefly, extraction solvent containing the internal standard is added to 20 uL of sample, followed by shaking and positive pressure filtering, and the resulting extracted sample is analyzed. The assay was validated based on accepted quality standards (e.g., CLIA and CLSI) for clinical assays. Results: The method is linear over a wide range of concentrations, 2.0-1500 µM, with LOD of 0.60 µM and LOQ of 2.0 µM. The precision of the assay was 4-10% across analytes. The method was also validated against reference laboratories via blinded split-sample analysis and demonstrated good agreement with accuracy: 89-95% relative to the external group mean. Conclusion: We have developed a method that is accurate, rapid, and reliable for routine clinical testing of patient sample BCAA, which is used in the diagnosis and management of maple syrup urine disease (MSUD). The assay also has desirable characteristics, such as short run time, small sample volume requirement, simple sample preparation without the need for derivatization, and high throughput.

Development of a high-throughput differential mobility separation-tandem mass spectrometry (DMS-MS/MS) method for clinical urine drug testing.

INTRODUCTION: Differential mobility separation (DMS) is an analytical technique used for rapid separation of ions and isomers based on gas phase mobility prior to entering a mass spectrometer for analysis. The entire DMS process is accomplished in fewer than 20 ms and can be used as a rapid alternative to chromatographic separation. OBJECTIVE: The primary objective was to evaluate the utility of DMS-tandem mass spectrometry (DMS-MS/MS) as a replacement for immunoassay-based clinical toxicology testing. METHODS: A sensitive DMS-MS/MS method was developed and validated for simultaneous identification of 33 drugs and metabolites in human urine samples. After DMS optimization, the method was validated and used to screen 56 clinical urine samples. These results were compared to results obtained by immunoassay. RESULTS: The DMS-MS/MS method achieved limits of detection ranging from 5 to 100 ng/mL. Moreover, the total analysis time was 2 min per sample. For the method performance evaluation, DMS-MS/MS results were compared with previously obtained urine toxicology immunoassay results. DMS-MS/MS showed higher sensitivity and identified 20% more drugs in urine, which were confirmed by LC-MS/MS. CONCLUSION: The DMS-MS/MS as applied in our lab demonstrated the capability for rapid drug screening and provided better analytical performance than immunoassay.

The Emerging Potential of Advanced Targeted Mass Spectrometry to Become a Routine Tool for Protein Quantification in Biomedical Research.

Mass spectrometry-based proteomics has become an indispensable tool for system-wide protein quantification in systems biology, biomedical research, and increasing for clinical applications. In particular, targeted mass spectrometry offers the most sensitive and reproducible quantitative detection of proteins, peptides and post-translational modifications of any currently applied mass spectrometry technique and is therefore ideally suited to generate high quality quantitative datasets. Despite these apparent advantages, targeted mass spectrometry is only slowly gaining popularity in academia and pharmaceutical industries, mainly due to the additional efforts in assay generation and manual data validation. However, with the increasing accumulation of mass spectrometry data, advances in deep learning spectral prediction for automated assay development, these obstacles can and will be considerably reduced in the near future. Here, we describe the latest technological developments in this field and discuss the emerging importance of targeted mass spectrometry for systems biology research and potential key roles in bridging biomedical discovery and clinical implementation.

Assessment of Mitochondrial Protein Composition and Purity by Mass Spectroscopy.

While the routine mitochondrial extraction and isolation protocols have not fundamentally changed since the introduction of density gradients, the approaches we use to examine the proteome have. The initial characterisation of mitochondrial proteomes was carried out using two-dimensional gel electrophoresis in 2001 and gel spot mass spectrometry have now largely been superseded as the throughput and sensitivity of commercial mass spectrometers increases. Whist many of these early studies established the components of the mitochondrial proteome, as gels were replaced by gel free approaches the numbers of confirmed components rapidly increased. In this chapter we present gel-based approaches for the separation and concentration of mitochondrial proteins for their characterization by mass spectrometry. We also describe two gel-free approaches which can be used to quantity the degree of contamination arising during the isolation of mitochondria. These approaches are equally suitable for studies comparing one treatment to another.

LC-MS lipidomics of renal biopsies for the diagnosis of Fabry disease.

INTRODUCTION: Lipidomics analysis or lipid profiling is a system-based analysis of all lipids in a sample to provide a comprehensive understanding of lipids within a biological system. In the last few years, lipidomics has made it possible to better understand the metabolic processes associated with several rare disorders and proved to be a powerful tool for their clinical investigation. Fabry disease is a rare X-linked lysosomal storage disorder (LSD) caused by a deficiency in α-galactosidase A (α-GAL A). This deficiency results in the progressive accumulation of glycosphingolipids, mostly globotriaosylceramide (Gb3), globotriaosylsphingosine (lyso-Gb3), as well as galabiosylceramide (Ga2) and their isoforms/analogs in the vascular endothelium, nerves, cardiomyocytes, renal glomerular podocytes, and biological fluids. OBJECTIVES: The primary objective of this study was to evaluate lipidomic signatures in renal biopsies to help understand variations in Fabry disease markers that could be used in future diagnostic tests. METHODS: Lipidomic analysis was performed by ultra-high pressure liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) on kidney biopsies that were left over after clinical pathology analysis to diagnose Fabry disease. RESULTS: We employed UHPLC-HRMS lipidomics analysis on the renal biopsy of a patient suspicious for Fabry disease. Our result confirmed α-GAL A enzyme activity declined in this patient since a Ga2-related lipid biomarker was substantially higher in the patient's renal tissue biopsy compared with two controls. This suggests this patient has a type of LSD that could be non-classical Fabry disease. CONCLUSION: This study shows that lipidomics analysis is a valuable tool for rare disorder diagnosis, which can be conducted on leftover tissue samples without disrupting normal patient care.

Rapid identification of plasmalogen molecular species using targeted multiplexed selected reaction monitoring mass spectrometry.

Plasmalogens (Pls) levels are reported to be altered in several neurological and metabolic diseases. Identification of sn-1 fatty alcohols and sn-2 fatty acids of different Pls species is necessary to determine the roles and mechanisms of action of Pls in different diseases. Previously, full-scan tandem mass spectrometry (MS/MS) was used for this purpose but is not effective for low-abundance Pls species. Recently, multiplexed selected reaction monitoring MS (SRM/MS) was found to be more selective and sensitive than conventional full-scan MS/MS for the identification of low-abundance compounds. In the present study, we developed a liquid chromatography (LC)-targeted multiplexed SRM/MS system for the identification and quantification of different Pls choline (Pls-PC) and Pls ethanolamine (Pls-PE) species. We determined five precursor-product ion transitions to identify sn-1 and sn-2 fragments of each Pls species. Consequently, sn-1 and sn-2 fatty acyl chains of 22 Pls-PC and 55 Pls-PE species were identified in mouse brain samples. Among them, some species had C20:0 and C20:1 fatty alcohols at the sn-1 position. For quantification of Pls species in mouse brain samples, a single SRM transition was employed. Thus, our results suggest that the LC-targeted multiplexed SRM/MS system is very sensitive for the identification and quantification of low-abundance lipids such as Pls, and is thus expected to make a significant contribution to basic and clinical research in this field in the future.

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

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