Bottom-up proteomic analysis of human adult cardiac tissue and isolated cardiomyocytes

• Published in: Journal of molecular and cellular cardiology Vol. 162; pp. 20 - 31
• Main Authors: Wojtkiewicz, Melinda, Berg Luecke, Linda, Castro, Chase, Burkovetskaya, Maria, Mesidor, Roneldine, Gundry, Rebekah L.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.01.2022
• Subjects: Cardiomyocyte isolation; Data independent acquisition; Humans; Myocytes, Cardiac - metabolism; Proteome - metabolism; Proteomics; Proteomics - methods; S-trap; Specimen Handling; Cardiomyocyte isolation; S-trap; Proteomics; Data independent acquisition
• ISSN: 0022-2828; 1095-8584
• DOI: 10.1016/j.yjmcc.2021.08.008

The heart is composed of multiple cell types, each with a specific function. Cell-type-specific approaches are necessary for defining the intricate molecular mechanisms underlying cardiac development, homeostasis, and pathology. While single-cell RNA-seq studies are beginning to define the chamber-specific cellular composition of the heart, our views of the proteome are more limited because most proteomics studies have utilized homogenized human cardiac tissue. To promote future cell-type specific analyses of the human heart, we describe the first method for cardiomyocyte isolation from cryopreserved human cardiac tissue followed by flow cytometry for purity assessment. We also describe a facile method for preparing isolated cardiomyocytes and whole cardiac tissue homogenate for bottom-up proteomic analyses. Prior experience in dissociating cardiac tissue or proteomics is not required to execute these methods. We compare different sample preparation workflows and analysis methods to demonstrate how these can impact the depth of proteome coverage achieved. We expect this how-to guide will serve as a starting point for investigators interested in general and cell-type-specific views of the cardiac proteome. [Display omitted] •Cardiomyocyte isolation from cryopreserved human cardiac tissue.•The first method for flow cytometry assessment of human adult cardiomyocytes.•Facile preparation method for bottom-up proteomic analyses of cardiac tissue.•Similarities and differences in cardiomyocytes from left and right ventricle.•How-to guide for general and cell-type-specific views of the cardiac proteome.