Tryptic Digestion is a necessity for big proteins, to overcome signal dilution due to multiple charge envelope and wide isotopic distribution when doing quantification by MRM. For smaller proteins, avoidance of digestion step could preserve peptidoforms information, and minimise variability. Thymosin beta-4 (TB4) is an X-linked gene product with cardioprotective properties. TB4 is a good candidate for developing a digestion-free LC-MS quantification, as it is not excessively big (approximately 5 kDa), very soluble, and is relatively abundant. We developed a digestion-free dilute-and-shoot method to quantify TB4 in plasma, then piloted the assay in heart failure (HF) cohort.
MRM for TB4 was developed empirically from synthetic standards, standard curve prepared by diluting in rabbit plasma. Plasma samples were spiked with heavy isotope internal standard, then “crashed” with acetonitrile. The assay was piloted in a nationwide heart failure cohort (n=438) with controls (n=219).
In HF patients compared to controls, plasma TB4 was significantly elevated [1265 (638–2146) ng/mL vs. 985 (421–1723) ng/mL, p=0.002]. Elevation seems to be primarily driven by women with heart failure with preserved ejection fraction (HFpEF) [1623(1040-2625) ng/ml]. Over the two years follow-up period, there were 60 deaths among patients with HF. Adjusted for NYHA class, N-terminal pro-B-type natriuretic peptide, age, and myocardial infarction, hazard ratio to all-cause mortality is significantly higher in women with elevated TB4 (1.668, p=0.036), but not in men (0.791, p=0.456) with HF. By also doing pairwise correlation of the TB4 measurements with biomarker information obtained from a multiplex proximity extension assay. We found that TB4 is strongly correlated (R > 0.7, p<<0.001) with a cluster of seven markers, six of which are either X-linked or regulated by sex-hormones. Given that the limited therapeutic options and poorly understood pathophysiology of HFpEF, our findings could lead to novel hypothesis.