Analysis of cardiac troponin proteoforms by top-down mass spectrometry

Abstract

The cardiac troponin complex, composed of three regulatory proteins (cTnI, cTnT, TnC), functions as the critical regulator of cardiac muscle contraction and relaxation. Myofilament protein-protein interactions are regulated by post-translational modifications (PTMs) to the protein constituents of this complex. Dysregulation of troponin PTMs, particularly phosphorylation, results in altered cardiac contractility. Altered PTMs and isoforms have been increasingly recognized as the molecular mechanisms underlying heart diseases. Therefore, it is essential to comprehensively analyze cardiac troponin proteoforms that arise from PTMs, alternative splicing, and sequence variations. In this chapter, we described two detailed protocols for the enrichment and purification of endogenous cardiac troponin proteoforms from cardiac tissue. Subsequently, mass spectrometry (MS)-based top-down proteomics utilizing online liquid chromatography (LC)/quadrupole time-of-flight (Q-TOF) MS for separation, profiling, and quantification of the troponins was demonstrated. Characterization of troponin amino acid sequence and the localization of PTMs were shown using Fourier-transform ion cyclotron resonance (FT-ICR) MS with electron capture dissociation (ECD) and collisionally activated dissociation (CAD). Furthermore, we described the use of MASH software, a comprehensive and free software package developed in our lab, for top-down proteomics data analysis. The methods we described can be applied for the analysis of troponin proteoforms in cardiac tissues, from animal models to human clinical samples, for heart disease.

Keywords: Cardiac troponin proteoforms; Phosphorylation; Protein extraction; Top-down proteomics.

Figure 1:. Characterization of intact cTn proteoforms by top-down mass spectrometry.

Schematic illustration of two workflows for intact cardiac troponin analysis: (1a) Myofilament subproteome extracted from cardiac tissue (pH-based); (1b) Online LC/MS proteoform profiling; (1c) Data analysis and relative proteoform quantification. (2a) Myofilament subproteome extracted from cardiac tissue (salt-based); (2b) Immunoaffinity purification of cTn complex; (2c) Offline MS/MS using CAD/ECD; (2d) Data analysis for characterization of cardiac troponin proteoforms.

Figure 2:. SDS-PAGE visualization of immunoaffinity purified cTn complex.

SDS-PAGE of immunoaffinity purified swine cTn complex from a cardiac tissue sample. M, molecular weight marker; LM, myofilament loading mixture; FT, flow through; E, elution fraction (1–5).

Figure 3:. Online LC/MS/MS of cardiac troponin I from human myofilament extracts using pH-based extraction.

A cTnI sequence map using online targeted CAD-based MS/MS for proteoform identification. From gel densitometry experiments (not depicted), approximately 100 ng of cTnI was loaded for online LC/MS/MS analysis using a Q-TOF instrument such as the Impact II.

Figure 4:. Online LC/MS of cardiac troponins from swine myofilament extracts using pH-based extraction.

(a) BPC, base peak chromatogram of swine myofilament-enriched extracts containing the cardiac troponins, separated by reverse-phase LC. (b) Online LC/MS spectra of cTnT and cTnI using an Impact II Q-TOF. The charge state envelope of cTnT and cTnI are depicted above. (c) Deconvoluted mass spectra of cTnT and cTnI, illustrating online LC/MS-based proteoform profiling. The red “p” represents phosphorylation.

Figure 5:. Offline MS/MS analysis of immunoaffinity purified cTnI using CAD and ECD.

ECD and CAD fragment ions and sequence map of a bis-phosphorylated swine cTnI proteoform with Met excision and N-terminal acetylation. A series of c ions including c₂₁ with no phosphorylation, c₂₂ with one phosphorylation, and c₂₃ with two phosphorylations, confirmed phosphorylation sites at Ser22 and Ser 23. The presence of acetylation was localized within the first six residues by a c₆ ion. The red “p” represents phosphorylation; Ac, acetylation.

Figure 6:. Quantification of relative proteoform abundance (cTnI) and total phosphorylation level (cTnT) using online LC-MS data.

(a) Relative proteoform abundance calculation using the proteoform intensities of cTnI, pcTnI, and ppcTnI obtained from a deconvoluted mass spectra of cTnI (Figure 4c). (b) Total phosphorylation level calculation using the relative proteoform abundance percentage of pcTnT obtained from a deconvoluted mass spectra of cTnT (Figure 4c).

Figure 7:. Offline MS/MS PTM characterization of cTnI using MASH software.

MASH software facilitates the characterization of PTMs and sequence variations by allowing users to add modifications to individual amino acids in a protein sequence. Without considering the presence of N-terminal acetylation, only 3 c ions and 45 z• ions could be identified from an offline ECD-based experiment of an immunoaffinity purified swine cTnI proteoform. With the addition of N-terminal acetylation, an additional 30 c ions were assigned.