Deciphering modifications in swine cardiac troponin I by top-down high-resolution tandem mass spectrometry

Abstract

Cardiac troponin I (cTnI) is an important regulatory protein in cardiac muscle, and its modification represents a key mechanism in the regulation of cardiac muscle contraction and relaxation. cTnI is often referred to as the "gold-standard" serum biomarker for diagnosing patients with acute cardiac injury since it is unique to the heart and released into the circulation following necrotic death of cardiac tissue. The swine (Sus scrofa) heart model is extremely valuable for cardiovascular research since the heart anatomy and coronary artery distribution of swine are almost identical to those of humans. Herein, we report a comprehensive characterization of the modifications in swine cTnI using top-down high-resolution tandem mass spectrometry in conjugation with immunoaffinity chromatography purification. High-resolution high accuracy mass spectrometry revealed that swine cTnI affinity purified from domestic pig hearts was N-terminally acetylated and phosphorylated. Electron capture disassociation is uniquely suited for localization of labile phosphorylations, which unambiguously identified Ser22/Ser23 as the only basally phosphorylated sites that are well-known to be regulated by protein kinase A and protein kinase C. Moreover, a combination of tandem mass spectrometry with sequence homology alignment effectively localized a single amino acid polymorphism, V116A, representing a novel genetic variant of swine cTnI. Overall, our studies demonstrated the unique power of top-down high-resolution tandem mass spectrometry in the characterization of protein modifications, including labile phosphorylation and unexpected sequence variants.

Figure 1. High-resolution MS analysis of intact cTnI purified from domestic pig hearts

(a) Broadband ESI/FTMS spectrum of desalted swine cTnI sample. (b) Expanded MS spectrum of swine cTnI from m/z 818 to m/z 868. (c) Narrow band FTMS spectrum of swine cTnI for the charge state 28⁺ precursor ions, showing its distribution in un-, mono- and bisphosphorylated forms. Insets: isotopically resolved molecular ions of un-, mono-, and bisphosphorylated cTnI (M²⁸⁺) with high accuracy molecular weight measurements. _pcTnI and _ppcTnI represent mono-(+79.95 Da) and bisphosphorylated (+159.92 Da) cTnI, respectively. Dashed arrow indicates the expected position for trisphosphorylated cTnI (_pppcTnI), which is not observed here. Circles represent the theoretical isotopic abundance distribution of the isotopomer peaks corresponding to the assigned mass. Calc’d, calculated most abundant mass; Expt’l, experimental most abundant mass. +H₃PO₄, non-covalent adduct of phosphoric acid (+98 Da).

Figure 2. Partial sequence alignment of cTnI from several mammalian species using a Clustal (2.0.11) algorithm

Sequences were downloaded from UnitProtKB/Swiss-Prot. Two splicing isoforms of pig cTnI sequences were aligned with cTnI sequences of bovine, human, mouse and rat. Numerals indicating amino acid positions are according to the full sequence with the initiating N-terminal Met. Amino acid differences between two swine cTnI isoforms were highlighted in rectangles.

Figure 3. ECD spectrum of isolated bisphosphorylated cTnI (_ppcTnI)

The product ion assignments were made according to the sequence A5X5T5_PIG with the removal of N-terminal Met and acetylation at the new terminus.

Figure 4. Key product ions from the ECD spectra for localization of swine cTnI bisphosphorylation sites

Solid arrow indicates the observed product ions; dashed arrow indicates the absence of the labeled product ions. Circles represent the theoretical isotopic abundance distribution of the isotopomer peaks corresponding to the assigned mass.

Figure 5. Representative product ions from the CAD spectrum of swine cTnI for localization of the mass discrepancy (−28 Da)

Product ions are assigned according to the sequence A5X5T5 with the removal of N-terminal Met and acetylation at the new terminus. Circles represent the theoretical isotopic abundance distribution of the isotopomer peaks corresponding to the assigned mass. Calc’d, calculated most abundant mass; Expt’l, experimental most abundant mass.

Figure 6. MS/MS fragmentation and product ion map from ECD and CAD spectra for (a) un- and (b) bisphosphorylated swine cTnI (_ppcTnI)

Fragments assignments were made to the swine cTnI (UnitProtKB/Swiss-Prot A5X5T5, TNNI3_pig) with the removal of N-terminal Met and acetylation at the new terminus. Bisphosphorylation sites of Ser22/23 were highlighted by circles. The fragmentation ions carrying the mass discrepancy (−28 Da) was indicated in dots. The potential amino acids containing the mass discrepancy (−28 Da) were highlighted in shades.