Assessment of protein extraction and digestion efficiency of well-established shotgun protocols for heart proteomics

Area of Science:

• Proteomics
• Cardiovascular Research
• Biochemistry

Background:

• Ischemic heart disease is a major global cause of mortality, necessitating a deeper understanding of its molecular underpinnings.
• Comprehensive proteomic analysis of cardiac tissue is crucial for elucidating disease mechanisms, yet optimal sample preparation methods remain underexplored.

Purpose of the Study:

• To compare the efficacy of three established shotgun proteomics sample preparation protocols for left ventricle (LV) tissue analysis.
• To evaluate protein extraction yield, protein identification numbers, and efficiency concerning protein abundance and protocol duration.

Main Methods:

• Three distinct protein digestion methods were assessed: in-solution digestion (ISD), on-pellet digestion (OPD), and on-filter digestion (OFD).
• Protein extraction utilized SDS-containing buffer for OPD/OFD and urea-containing buffer for ISD.
• Dimethyl labeling was employed for relative protein quantitation, and an optimized one-step reduction/alkylation reaction was used for ISD and OPD.

Main Results:

• ISD and OFD protocols extracted significantly higher protein quantities from equivalent amounts of LV tissue compared to OPD.
• ISD identified a greater number of proteins than both OFD and OPD, demonstrating superior performance in protein coverage.
• ISD proved more effective in identifying low-abundance proteins and offered a higher protein identification rate relative to its duration.

Conclusions:

• The optimized in-solution digestion (ISD) protocol is the most suitable method for heart proteomics, outperforming on-filter digestion (OFD) and on-pellet digestion (OPD).
• ISD provides enhanced efficiency for comprehensive cardiac proteome analysis, particularly for low-abundance protein identification.