TY - JOUR
T1 - Bottom-up proteomic analysis of human adult cardiac tissue and isolated cardiomyocytes
AU - Wojtkiewicz, Melinda
AU - Berg Luecke, Linda
AU - Castro, Chase
AU - Burkovetskaya, Maria
AU - Mesidor, Roneldine
AU - Gundry, Rebekah L.
N1 - Funding Information:
This work was supported by the National Institutes of Health [ R01-HL134010 , R01- HL126785 ; R35- HL155460 to R.L.G.], American Heart Association [ 20PRE35200049 to L.B.L.]. LBL is a member of the MCW-MSTP which is partially supported by a T32 grant from NIGMS, GM080202. Funding sources were not involved in study design, data collection, interpretation, analysis or publication. Flow cytometry was performed using instrumentation in the UNMC Flow Cytometry Research Facility, administrated through the Office of the Vice Chancellor for Research, and supported by state funds from the Nebraska Research Initiative (NRI) and The Fred and Pamela Buffett Cancer Center's National Cancer Institute Cancer Support Grant. Major instrumentation has been provided by the Office of the Vice Chancellor for Research, The University of Nebraska Foundation, the Nebraska Banker's Fund, and by the NIH-NCRR Shared Instrument Program.
Publisher Copyright:
© 2021 The Authors
PY - 2022/1
Y1 - 2022/1
N2 - 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.
AB - 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.
KW - Cardiomyocyte isolation
KW - Data independent acquisition
KW - Proteomics
KW - S-trap
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U2 - 10.1016/j.yjmcc.2021.08.008
DO - 10.1016/j.yjmcc.2021.08.008
M3 - Article
C2 - 34437879
AN - SCOPUS:85114750482
VL - 162
SP - 20
EP - 31
JO - Journal of Molecular and Cellular Cardiology
JF - Journal of Molecular and Cellular Cardiology
SN - 0022-2828
ER -