Project description:The data presented here describes the use of targeted proteomic assays to quantify potential biomarkers of Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) sensitivity in lung adenocarcinoma and is related to the research article: "Quantitative targeted proteomic analysis of potential markers of tyrosine kinase inhibitor (TKI) sensitivity in EGFR mutated lung adenocarcinoma" . This article describes the data associated with liquid chromatography coupled to multiple reaction monitoring (LC-MRM) method development which includes selection of an optimal transition list, retention time prediction and building of reverse calibration curves. Sample preparation and optimization which includes phosphotyrosine peptide enrichment via a combination of pan-phosphotyrosine antibodies is described. The dataset also consists of figures, tables and Excel files describing the quantitative results of testing these optimized methods in two lung adenocarcinoma cell lines with EGFR mutations.
Project description:Heterotopic ossification (HO) is a significant problem for wounded warriors surviving high-energy blast injuries; however, currently, there is no biomarker panel capable of globally characterizing, diagnosing, and monitoring HO progression. The aim of this study was to identify biomarkers for HO using proteomic techniques and blood serum.Isobaric tags for relative and absolute quantitation (iTRAQ) was used to generate a semi-quantitative global proteomics survey of serum from patients with and without heterotopic ossification. Leveraging the iTRAQ data, a targeted selection reaction monitoring mass spectrometry (SRM-MS) assay was developed for 10 protein candidates: alkaline phosphatase, osteocalcin, alpha-2 type I collagen, collagen alpha-1(V) chain isoform 2 preprotein, bone sialoprotein 2, phosphatidate phosphatase LPIN2, osteomodulin, protein phosphatase 1J, and RRP12-like protein.The proteomic survey of serum from both healthy and disease patients includes 1220 proteins and was enriched for proteins involved in the response to elevated platelet Ca+2, wound healing, and extracellular matrix organization. Proteolytic peptides from three of the ten SRM-MS proteins, osteocalcin preprotein, osteomodulin precursor, and collagen alpha-1(v) chain isoform 2 preprotein from serum, are potential clinical biomarkers for HO.This study is the first reported SRM-MS analysis of serum from individuals with and without heterotopic ossification, and differences in the serum proteomic profile between healthy and diseased subjects were identified. Furthermore, our results indicate that normal wound healing signals can impact the ability to identify biomarkers, and a multi-protein panel assay, including osteocalcin preproprotein, osteomodulin precursor, and collagen alpha-1(v) chain isoform 2 preprotein, may provide a solution for HO detection and monitoring.
Project description:There is an increasing interest in the use of dried blood spot (DBS) sampling and multiple reaction monitoring in proteomics. Although several groups have explored the utility of DBS by focusing on protein detection, the reproducibility of the approach and whether it can be used for biomarker discovery in high throughput studies is yet to be determined. We assessed the reproducibility of multiplexed targeted protein measurements in DBS compared to serum. Eighty-two medium to high abundance proteins were monitored in a number of technical and biological replicates. Importantly, as part of the data analysis, several statistical quality control approaches were evaluated to detect inaccurate transitions. After implementing statistical quality control measures, the median CV on the original scale for all detected peptides in DBS was 13.2% and in Serum 8.8%. We also found a strong correlation (r?=?0.72) between relative peptide abundance measured in DBS and serum. The combination of minimally invasive sample collection with a highly specific and sensitive mass spectrometry (MS) technique allows for targeted quantification of multiple proteins in a single MS run. This approach has the potential to fundamentally change clinical proteomics and personalized medicine by facilitating large-scale studies.
Project description:Associating changes in protein levels with the onset of cancer has been widely investigated to identify clinically relevant diagnostic biomarkers. In the present study, we analyzed sera from 205 patients recruited in the United States and Egypt for biomarker discovery using label-free proteomic analysis by LC-MS/MS. We performed untargeted proteomic analysis of sera to identify candidate proteins with statistically significant differences between hepatocellular carcinoma (HCC) and patients with liver cirrhosis. We further evaluated the significance of 101 proteins in sera from the same 205 patients through targeted quantitation by MRM on a triple quadrupole mass spectrometer. This led to the identification of 21 candidate protein biomarkers that were significantly altered in both the United States and Egyptian cohorts. Among the 21 candidates, ten were previously reported as HCC-associated proteins (eight exhibiting consistent trends with our observation), whereas 11 are new candidates discovered by this study. Pathway analysis based on the significant proteins reveals upregulation of the complement and coagulation cascades pathway and downregulation of the antigen processing and presentation pathway in HCC cases versus patients with liver cirrhosis. The results of this study demonstrate the power of combining untargeted and targeted quantitation methods for a comprehensive serum proteomic analysis, to evaluate changes in protein levels and discover novel diagnostic biomarkers. All MS data have been deposited in the ProteomeXchange with identifier PXD001171 (http://proteomecentral.proteomexchange.org/dataset/PXD001171).
Project description:Multiple reaction monitoring mass spectrometry (MRM-MS) is a targeted analysis method that has been increasingly viewed as an avenue to explore proteomes with unprecedented sensitivity and throughput. We have developed a software tool, called MaRiMba, to automate the creation of explicitly defined MRM transition lists required to program triple quadrupole mass spectrometers in such analyses. MaRiMba creates MRM transition lists from downloaded or custom-built spectral libraries, restricts output to specified proteins or peptides, and filters based on precursor peptide and product ion properties. MaRiMba can also create MRM lists containing corresponding transitions for isotopically heavy peptides, for which the precursor and product ions are adjusted according to user specifications. This open-source application is operated through a graphical user interface incorporated into the Trans-Proteomic Pipeline, and it outputs the final MRM list to a text file for upload to MS instruments. To illustrate the use of MaRiMba, we used the tool to design and execute an MRM-MS experiment in which we targeted the proteins of a well-defined and previously published standard mixture.
Project description:Available blood assays for venous thromboembolism (VTE) suffer from diminished specificity. Compared with single marker tests, such as D-dimer, a multi-marker strategy may improve diagnostic ability. We used direct mass spectrometry (MS) analysis of serum from patients with VTE to determine whether protein expression profiles would predict diagnosis.We developed a direct MS and computational approach to the proteomic analysis of serum. Using this new method, we analyzed serum from inpatients undergoing radiographic evaluation for VTE. In a balanced cohort of 76 patients, a neural network-based prediction model was built using a training subset of the cohort to first identify proteomic patterns of VTE. The proteomic patterns were then validated in a separate group of patients within the cohort. The model yielded a sensitivity of 68% and specificity of 89%, which exceeded the specificity of D-dimer assay tested by latex agglutination, ELISA, and immunoturbimetric methods (sensitivity/specificity of 63.2%/60.5%, 97.4%/21.1%, 97.4%/15.8%, respectively). We validated differences in protein expression between patients with and without VTE using more traditional gel-based analysis of the same serum samples.Protein expression analysis of serum using direct MS demonstrates potential diagnostic utility for VTE. This pilot study is the first such direct MS study to be applied to a cardiovascular disease. Differences in protein expression were identified and subsequently validated in a separate group of patients. The findings in this initial cohort can be evaluated in other independent cohorts, including patients with inflammatory conditions and chronic (but not acute) VTE, for the diagnosis of VTE.
Project description:Personalized cancer medicine requires the development of tumor-specific biomarkers to optimize selection of targeted therapies and to better assess response to therapy. Current efforts in several tumor types have shown that patients in whom circulating tumor cells (CTCs) are detected have an inferior prognosis relative to those in whom CTCs are not detected and that the elimination or decrease of CTCs following treatment is associated with improved clinical outcomes. Technological advances in the detection, isolation, capture, and characterization of CTCs from phlebotomy samples obtained in a routine clinical practice setting have enabled the evaluation of different CTC biomarkers. Unmet needs in cancer diagnosis and treatment where CTC biomarkers have been studied include determining prognosis, assessing the effects of treatment, and as a source of tumor for the biologic identification and characterization of determinants to predict sensitivity to one form of treatment versus another and to understand mechanisms of treatment resistance.At present, there is no single definition of a CTC and no single CTC "biomarker." Rather, multiple assays (tests) are in development for CTC biomarkers. However, before the role of any biomarker in medical decision making can be determined, it is essential that the assays used to measure the biomarker are analytically validated in a sequence of trials to generate the evidence to support the biomarker's use in the given context of use. It is against this background that this review focuses on the process of developing CTC biomarker assays, with the objective of outlining the necessary steps to qualify specific CTC tests for medical decision making in clinical practice or drug development. The potential for point-of-care tests is clear.
Project description:<h4>Background</h4>Targeted proteomics, which involves quantitative analysis of targeted proteins using selected reaction monitoring (SRM) mass spectrometry, has emerged as a new methodology for discovery of clinical biomarkers. In this study, we used targeted serum proteomics to identify circulating biomarkers for prediction of disease activity and organ involvement in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV).<h4>Methods</h4>A large-scale SRM assay targeting 135 biomarker candidates was established using a triple-quadrupole mass spectrometer coupled with nanoflow liquid chromatography. Target proteins in serum samples from patients in the active and remission (6 months after treatment) stages were quantified using the established assays. Identified marker candidates were further validated by enzyme-linked immunosorbent assay using serum samples (n?=?169) collected in a large-cohort Japanese study (the RemIT-JAV-RPGN study).<h4>Results</h4>Our proteomic analysis identified the following proteins as biomarkers for discriminating patients with highly active AAV from those in remission or healthy control subjects: tenascin C (TNC), C-reactive protein (CRP), tissue inhibitor of metalloproteinase 1 (TIMP1), leucine-rich alpha-2-glycoprotein 1, S100A8/A9, CD93, matrix metalloproteinase 9, and transketolase (TKT). Of these, TIMP1 was the best-performing marker of disease activity, allowing distinction between mildly active AAV and remission. Moreover, in contrast to CRP, serum levels of TIMP1 in patients with active AAV were significantly higher than those in patients with infectious diseases. The serum levels of TKT and CD93 were higher in patients with renal involvement than in those without, and they predicted kidney outcome. The level of circulating TNC was elevated significantly in patients with lung infiltration. AAV severity was associated with markers reflecting organ involvement (TKT, CD93, and TNC) rather than inflammation. The eight markers and myeloperoxidase (MPO)-ANCA were clustered into three groups: MPO-ANCA, renal involvement (TKT and CD93), and inflammation (the other six markers).<h4>Conclusions</h4>We have identified promising biomarkers of disease activity, disease severity, and organ involvement in AAV with a targeted proteomics approach using serum samples obtained from a large-cohort Japanese study. Especially, our analysis demonstrated the effectiveness of TIMP1 as a marker of AAV activity. In addition, we identified TKT and CD93 as novel markers for evaluation of renal involvement and kidney outcome in AAV.
Project description:Sorafenib is the only standard treatment for unresectable hepatocellular carcinoma (HCC), but it provides modest survival benefits over placebo, necessitating predictive biomarkers of the response to sorafenib. Serum samples were obtained from 115 consecutive patients with HCC before sorafenib treatment and analyzed by multiple reaction monitoring-mass spectrometry (MRM-MS) and ELISA to quantify candidate biomarkers. We verified a triple-marker panel to be predictive of the response to sorafenib by MRM-MS, comprising CD5 antigen-like (CD5L), immunoglobulin J (IGJ), and galectin-3-binding protein (LGALS3BP), in HCC patients. This panel was a significant predictor (AUROC > 0.950) of the response to sorafenib treatment, having the best cut-off value (0.4) by multivariate analysis. In the training set, patients who exceeded this cut-off value had significantly better overall survival (median, 21.4 months) than those with lower values (median, 8.6 months; <i>p</i> = 0.001). Further, a value that was lower than this cutoff was an independent predictor of poor overall survival [hazard ratio (HR), 2.728; 95% confidence interval (CI), 1.312-5.672; <i>p</i> = 0.007] and remained an independent predictive factor of rapid progression (HR, 2.631; 95% CI, 1.448-4.780; <i>p</i> = 0.002). When applied to the independent validation set, levels of the cut-off value for triple-marker panel maintained their prognostic value for poor clinical outcomes. On the contrast, the triple-marker panel was not a prognostic factor for patients who were treated with transarterial chemoembolization (TACE). The discriminatory signature of a triple-marker panel provides new insights into targeted proteomic biomarkers for individualized sorafenib therapy.
Project description:Because of the growing impact of late onset cognitive loss, considerable effort has been directed toward the development of improved diagnostic techniques for Alzheimer's disease (AD) that may pave the way for earlier (and more effective) therapeutic efforts. Serum-based biomarkers are the least expensive and invasive modality for screening and routine monitoring. We systematically reviewed the literature to assemble a list of serum biomarkers relevant to AD. In parallel, we conducted a proteomic LC-MS/MS analysis of serum collected from neurologically normal subjects and subjects with mild cognitive impairment (MCI) and early AD (n = 6 in all). Complement C3 and alpha-2-macroglobulin were identified from both the literature review and our proteomic screen for further validation. For these two candidates, ELISA was performed on serum collected from a small independent cohort of subjects for longitudinal analysis. Serum was serially collected from neurologically normal subjects (n = 5) and subjects with MCI who were subsequently followed for a period of two years (n = 5) and regrouped into stable MCI and progressive MCI or AD (n = 6). The ability of each marker to predict which subjects with MCI would progress to dementia and which would remain cognitively stable was assessed. Patients with probable cerebral amyloid angiopathy were also identified (n = 3). This preliminary analysis tested the most-promising serum protein biomarkers for AD and we concluded that none are yet ready for use in the clinical diagnosis and management of dementia. However, a more thorough assessment in longitudinal studies with higher statistical power is warranted.