Simultaneous Quantification of Protein Expression and Modifications by Top-down Targeted Proteomics: A Case of the Sarcomeric Subproteome.
Ziqing LinLiming WeiWenxuan CaiYanlong ZhuTrisha TucholskiStanford D MitchellWei GuoStephen P FordGary M DiffeeYing GePublished in: Molecular & cellular proteomics : MCP (2018)
Determining changes in protein expression and post-translational modifications (PTMs) is crucial for elucidating cellular signal transduction and disease mechanisms. Conventional antibody-based approaches have inherent problems such as the limited availability of high-quality antibodies and batch-to-batch variation. Top-down mass spectrometry (MS)-based proteomics has emerged as the most powerful method for characterization and quantification of protein modifications. Nevertheless, robust methods to simultaneously determine changes in protein expression and PTMs remain lacking. Herein, we have developed a straightforward and robust top-down liquid chromatography (LC)/MS-based targeted proteomics platform for simultaneous quantification of protein expression and PTMs with high throughput and high reproducibility. We employed this method to analyze the sarcomeric subproteome from various muscle types of different species, which successfully revealed skeletal muscle heterogeneity and cardiac developmental changes in sarcomeric protein isoform expression and PTMs. As demonstrated, this targeted top-down proteomics platform offers an excellent 'antibody-independent' alternative for the accurate quantification of sarcomeric protein expression and PTMs concurrently in complex mixtures, which is generally applicable to different species and various tissue types.
Keyphrases
- mass spectrometry
- liquid chromatography
- high throughput
- hypertrophic cardiomyopathy
- skeletal muscle
- high resolution
- capillary electrophoresis
- high resolution mass spectrometry
- gas chromatography
- high performance liquid chromatography
- single cell
- cancer therapy
- tandem mass spectrometry
- left ventricular
- label free
- mental health
- poor prognosis
- binding protein
- heart failure
- protein protein
- insulin resistance
- amino acid
- long non coding rna
- adipose tissue