Researchers have developed a simplified extraction method that reduced the amount of cardiac tissue needed to analyze phospholamban from 10 mg to 1 mg. The method maintained similar analytical performance despite using one-tenth of the tissue, according to findings published in the Journal of the American Society for Mass Spectrometry.
Phospholamban is a small membrane protein that helps control calcium movement and contraction in the heart. Changes in the protein and its chemical modifications have been associated with heart failure and inherited cardiomyopathies.
Studying phospholamban can be difficult because membrane proteins are poorly soluble and often present at low levels. Conventional preparation methods also involve several steps that can cause protein loss, making them unsuitable for small tissue samples.
The researchers developed a one-pot method to simplify this process using Azo, a surfactant that dissolves membrane proteins and can be broken down with ultraviolet light before mass spectrometry.
The team tested the method using 1 mg pieces of left ventricular tissue from a nonfailing adult donor heart. The tissue was placed directly in an Azo-containing buffer and treated with sonication to improve protein extraction. The method eliminated the cryopulverization and cytosolic protein depletion steps used in the previous workflow.
Following extraction, the researchers analyzed intact proteins using liquid chromatography-tandem mass spectrometry. Examining intact proteins allowed them to distinguish proteoforms – different forms of the same protein created by chemical modifications or genetic variation.
The one-pot method produced approximately 30 to 60 µg of total protein from 1 mg of cardiac tissue. It consistently detected unmodified, phosphorylated, and palmitoylated forms of phospholamban across three replicate experiments.
Measurements of phospholamban phosphorylation were comparable with those obtained using the conventional 10 mg method. Both approaches also achieved 71 percent sequence coverage for the protein carrying two phosphate groups. The researchers identified the phosphorylation sites as Ser16 and Thr17.
The study shows that detailed analysis of intact cardiac proteins may be possible with tissue quantities similar to those obtained through biopsy. This could support future studies of early molecular changes in cardiovascular disease or responses to treatment while preserving limited clinical specimens.
However, the method was evaluated using tissue from one nonfailing donor and focused only on phospholamban. It was not used to examine the full cardiac proteome or tested in patients with cardiovascular disease. The palmitoylated form was identified by its intact mass and separation pattern rather than confirmed by targeted tandem mass spectrometry in this study.
Further research is needed to assess the method in clinical biopsy samples, diseased tissue, and other cardiac proteins.
Newsletters
Receive the latest analytical science news, personalities, education, and career development – weekly to your inbox.
