Dataset Information

Data-independent acquisition mass spectrometry to quantify protein levels in FFPE tumor biopsies for molecular diagnostics

ABSTRACT: Mass spectrometry-based protein quantitation is currently used to measure therapeutically relevant protein biomarkers in CAP/CLIA setting to predict likely responses of known therapies. Selected reaction monitoring (SRM) is the method of choice due to its outstanding analytical performance. However, data-independent acquisition (DIA) is now emerging as a proteome-scale clinical assay. We evaluated the ability of DIA to profile the patient-specific proteomes of sample-limited tumor biopsies and to quantify proteins of interest in a targeted fashion using formalin-fixed, paraffin-embedded (FFPE) tumor biopsies (n=12) selected from our clinical laboratory. DIA analysis on the tumor biopsies provided 3,713 quantifiable proteins including actionable biomarkers currently in clinical use, successfully separated two gastric cancers from colorectal cancer specimen solely based on global proteomic profiles, and identified subtype-specific proteins with prognostic or diagnostic value. We demonstrate the potential use of DIA-based quantitation to inform therapeutic decision-making using TUBB3, for which clinical cut-off expression levels have been established by SRM. Comparative analysis of DIA-based proteomic profiles and mRNA expression levels found positively and negatively correlated protein-gene pairs, a finding consistent with previously reported results from fresh-frozen tumor tissues.

ORGANISM(S): Homo Sapiens

SUBMITTER: Steve Sweet

PROVIDER: PXD010934 | panorama | Tue Oct 22 00:00:00 BST 2019

REPOSITORIES: PanoramaPublic

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Data-Independent Acquisition Mass Spectrometry To Quantify Protein Levels in FFPE Tumor Biopsies for Molecular Diagnostics.

Kim Yeoun Jin YJ Sweet Steve M M SMM Egertson Jarrett D JD Sedgewick Andrew J AJ Woo Sunghee S Liao Wei-Li WL Merrihew Gennifer E GE Searle Brian C BC Vaske Charlie C Heaton Robert R MacCoss Michael J MJ Hembrough Todd T

Journal of proteome research 20181212 1

Mass spectrometry-based protein quantitation is currently used to measure therapeutically relevant protein biomarkers in CAP/CLIA setting to predict likely responses of known therapies. Selected reaction monitoring (SRM) is the method of choice due to its outstanding analytical performance. However, data-independent acquisition (DIA) is now emerging as a proteome-scale clinical assay. We evaluated the ability of DIA to profile the patient-specific proteomes of sample-limited tumor biopsies and t ...[more]

PMID: 30481034

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Project description:Although hepatocellular carcinoma (HCC) has been subjected to continuous investigation and its symptoms are well-known, early stage diagnosis of this disease remains difficult and the survival rate after diagnosis is typically very low (3−5%). Early and accurate detection of metabolic changes in the sera of patients with liver cirrhosis can help improve the prognosis of HCC and lead to a better understanding of its mechanism at the molecular level, thus providing patients with in-time treatment of the disease. In this study, we compared metabolite levels in sera of 40 HCC patients and 49 cirrhosis patients from Egypt by using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometer (UPLC-QTOF MS). Following data preprocessing, the most relevant ions in distinguishing HCC cases from cirrhotic controls are selected by statistical methods. Putative metabolite identifications for these ions are obtained through mass-based database search. The identities of some of the putative identifications are verified by comparing their MS/MS fragmentation patterns and retention times with those from authentic compounds. Finally, the serum samples are reanalyzed for quantitation of selected metabolites as candidate biomarkers of HCC. This quantitation was performed using isotope dilution by selected reaction monitoring (SRM) on a triple quadrupole linear ion trap (QqQLIT) coupled to UPLC. Statistical analysis of the UPLC-QTOF data identified 274 monoisotopic ion masses with statistically significant differences in ion intensities between HCC cases and cirrhotic controls. Putative identifications were obtained for 158 ions by mass based search against databases. We verified the identities of selected putative identifications including glycholic acid (GCA), glycodeoxycholic acid (GDCA), 3β, 6β-dihydroxy-5β-cholan-24-oic acid, oleoyl carnitine, and Phe-Phe. SRM-based quantitation confirmed significant differences between HCC and cirrhotic controls in metabolite levels of bile acid metabolites, long chain carnitines and small peptide. Our study provides useful insight into appropriate experimental design and computational methods for serum biomarker discovery using LC−MS/MS based metabolomics. This study has led to the identification of candidate biomarkers with significant changes in metabolite levels between HCC cases and cirrhotic controls. This is the first MS-based metabolic biomarker discovery study on Egyptian subjects that led to the identification of candidate metabolites that discriminate early stage HCC from patients with liver cirrhosis.

Dataset Information

Data-independent acquisition mass spectrometry to quantify protein levels in FFPE tumor biopsies for molecular diagnostics

Publications

Data-Independent Acquisition Mass Spectrometry To Quantify Protein Levels in FFPE Tumor Biopsies for Molecular Diagnostics.

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