Top-down quantitative proteomics identified phosphorylation of cardiac troponin I as a candidate biomarker for chronic heart failure

Abstract

The rapid increase in the prevalence of chronic heart failure (CHF) worldwide underscores an urgent need to identify biomarkers for the early detection of CHF. Post-translational modifications (PTMs) are associated with many critical signaling events during disease progression and thus offer a plethora of candidate biomarkers. We have employed a top-down quantitative proteomics methodology for comprehensive assessment of PTMs in whole proteins extracted from normal and diseased tissues. We systematically analyzed 36 clinical human heart tissue samples and identified phosphorylation of cardiac troponin I (cTnI) as a candidate biomarker for CHF. The relative percentages of the total phosphorylated cTnI forms over the entire cTnI populations (%P(total)) were 56.4 ± 3.5%, 36.9 ± 1.6%, 6.1 ± 2.4%, and 1.0 ± 0.6% for postmortem hearts with normal cardiac function (n = 7), early stage of mild hypertrophy (n = 5), severe hypertrophy/dilation (n = 4), and end-stage CHF (n = 6), respectively. In fresh transplant samples, the %P(total) of cTnI from nonfailing donor (n = 4), and end-stage failing hearts (n = 10) were 49.5 ± 5.9% and 18.8 ± 2.9%, respectively. Top-down MS with electron capture dissociation unequivocally localized the altered phosphorylation sites to Ser22/23 and determined the order of phosphorylation/dephosphorylation. This study represents the first clinical application of top-down MS-based quantitative proteomics for biomarker discovery from tissues, highlighting the potential of PTMs as disease biomarkers.

Fig. 1

Schematic representation of top-down quantitative proteomics methodology featuring affinity-chromatography and high-resolution MS for comprehensive analysis of PTMs in whole proteins extracted from normal and diseased tissues.

Fig. 2

Representative high-resolution ESI/FTMS spectra of whole cTnI (M²⁸⁺) purified from (A) postmortem and (B) fresh transplant human heart samples. A, i) NOR2; ii) HYP3; and B, i) DOR1; (ii) ICM3. Roman numerals (II–IV) indicate three different C-terminally truncated isoforms of N-terminally acetylated cTnI (II, 1–207; III, 1–206, IV, 1–205). Subscripts p and pp stand for mono and bisphosphorylation. +H₃PO₄, non-covalent adduct of phosphoric acid. Asterisks indicate co-purified minor cTnT related products. NOR, control heart with normal function; HYP, mild hypertrophy; DOR, donor heart; ICM, ischemic cardiomyopathy. Clinical details of the myocardial tissue samples were shown in Table S1–2.

Fig. 3

Correlation between the cTnI phosphorylation level and heart disease phenotypes. Representative cases from (A) postmortem heart samples: i–ii) NOR2,3; iii–iv) HYP2,3; v–vi) SHD1,2; vii–viii) CHF2,3; and (B) fresh transplant heart samples, i–ii) DOR1,4; iii–iv) ICM3, 6; v–vi) DCM 3,4. Clinical details of the myocardial tissue samples were shown in Supplemental Table S1–2.

Fig. 4

Relative quantification of cTnI phosphorylation in normal and diseased human heart samples. (A) postmortem (NOR, control heart with normal function; HYP, mild hypertrophy, SHD, severe hypertrophy/dilation; CHF, congestive heart failure), and (B) fresh transplant (DOR, donor heart; ICM/DCM, ischemic/dilated cardiomyopathy) samples. i) Total phosphorylated cTnI percentage (%P_total); ii) mono-phosphorylated cTnI percentage (%P_mono); and iii) bis-phosphorylated cTnI percentage (%P_bis) over the entire cTnI populations. Box, median and interquartile range (25%, 75%); whiskers, minimum and maximum values. *p<0.05.

Fig. 5

Mapping cTnI phosphorylation sites by top-down MS with ECD. Fragmentation maps of _pcTnI in (A) normal, (B) hypertrophic heart samples and (C) _ppcTnI in normal heart samples. The identified phosphorylation site(s) of Ser22 in _pcTnI and Ser22/23 in _ppcTnI are highlighted in circles.

Fig. 6

Relative quantification of cTnI degradation levels in normal and diseased heart samples. NOR, normal controls, HYP, mild hypertrophy, SHD, severe hypertrophy/dilation, and CHF, congestive heart failure. (a) Total degraded cTnI percentage (%D_total); (b–f) percentages of degradation products cTnI(II), (III), (IV), [Y28–205K], [Y28–206K], over the entire cTnI populations, respectively. Calculated p values were not found to be statistically significant