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Demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins.

ABSTRACT: Multiple reaction monitoring (MRM) mass spectrometry has been successfully applied to monitor targeted proteins in biological specimens, raising the possibility that assays could be configured to measure all human proteins. We report the results of a pilot study designed to test the feasibility of a large-scale, international effort for MRM assay generation. We have configured, validated across three laboratories and made publicly available as a resource to the community 645 novel MRM assays representing 319 proteins expressed in human breast cancer. Assays were multiplexed in groups of >150 peptides and deployed to quantify endogenous analytes in a panel of breast cancer-related cell lines. The median assay precision was 5.4%, with high interlaboratory correlation (R(2) > 0.96). Peptide measurements in breast cancer cell lines were able to discriminate among molecular subtypes and identify genome-driven changes in the cancer proteome. These results establish the feasibility of a large-scale effort to develop an MRM assay resource.

SUBMITTER: Kennedy JJ

PROVIDER: S-EPMC3922286 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins.

Kennedy Jacob J JJ Abbatiello Susan E SE Kim Kyunggon K Yan Ping P Whiteaker Jeffrey R JR Lin Chenwei C Kim Jun Seok JS Zhang Yuzheng Y Wang Xianlong X Ivey Richard G RG Zhao Lei L Min Hophil H Lee Youngju Y Yu Myeong-Hee MH Yang Eun Gyeong EG Lee Cheolju C Wang Pei P Rodriguez Henry H Kim Youngsoo Y Carr Steven A SA Paulovich Amanda G AG

Nature methods 20131208 2

Multiple reaction monitoring (MRM) mass spectrometry has been successfully applied to monitor targeted proteins in biological specimens, raising the possibility that assays could be configured to measure all human proteins. We report the results of a pilot study designed to test the feasibility of a large-scale, international effort for MRM assay generation. We have configured, validated across three laboratories and made publicly available as a resource to the community 645 novel MRM assays rep ...[more]

PMID: 24317253

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Project description:BackgroundDuring aging, there is a decreased ability to maintain skeletal muscle mass and function (sarcopenia). Such changes in skeletal muscle are also co-morbidities of diseases including cancer, congestive heart failure and chronic obstructive pulmonary disease. The loss of muscle mass results in decreased strength and exercise tolerance and reduced ability to perform daily activities. Pharmacological agents addressing these pathologies could have significant clinical impact, but their identification requires understanding of mechanisms driving myotube formation (myogenesis) and atrophy and provision of relevant assays. The aim of this study was to develop robust in vitro methods to study human myogenesis.MethodsSatellite cells were isolated by digestion of post-mortem skeletal muscle and selection using anti-CD56 MicroBeads. CD56(+) cell-derived myotubes were quantified by high content imaging of myosin heavy chains. TaqMan-polymerase chain reaction arrays were used to quantify expression of 41 selected genes during differentiation. The effects of activin receptor agonists and tumour necrosis factor alpha (TNFα) on myogenesis and gene expression were characterised.ResultsLarge-scale isolation of CD56(+) cells enabled development of a quantitative myogenesis assay with maximal myotube formation 3 days after initiating differentiation. Gene expression analysis demonstrated expression of 19 genes changed substantially during myogenesis. TNFα and activin receptor agonists inhibited myogenesis and downregulated gene expression of muscle transcription factors, structural components and markers of oxidative phenotype, but only TNFα increased expression of pro-inflammatory markers.ConclusionsWe have developed methods for large-scale isolation of satellite cells from muscle and quantitative assays for studying human myogenesis. These systems may prove useful as part of a screening cascade designed to identify therapeutic agents for improving muscle function.

Dataset Information

Demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins.

Publications

Demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins.

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