In vivo phosphorylation site mapping in mouse cardiac troponin I by high resolution top-down electron capture dissociation mass spectrometry: Ser22/23 are the only sites basally phosphorylated

Abstract

Cardiac troponin I (cTnI) is the inhibitory subunit of cardiac troponin, a key myofilament regulatory protein complex located on the thin filaments of the contractile apparatus. cTnI is uniquely specific for the heart and is widely used in clinics as a serum biomarker for cardiac injury. Phosphorylation of cTnI plays a critical role in modulating cardiac function. cTnI is known to be regulated by protein kinase A and protein kinase C at five sites, Ser22/Ser23, Ser42/44, and Thr143, primarily based on results from in vitro phosphorylation assays by the specific kinase(s). However, a comprehensive characterization of phosphorylation of mouse cTnI occurring in vivo has been lacking. Herein, we have employed top-down mass spectrometry (MS) methodology with electron capture dissociation for precise mapping of in vivo phosphorylation sites of cTnI affinity purified from wild-type and transgenic mouse hearts. As demonstrated, top-down MS (analysis of intact proteins) is an extremely valuable technology for global characterization of labile phosphorylation occurring in vivo without a priori knowledge. Our top-down MS data unambiguously identified Ser22/23 as the only two sites basally phosphorylated in wild-type mouse cTnI with full sequence coverage, which was confirmed by the lack of phosphorylation in cTnI-Ala(2) transgenic mice where Ser22/23 in cTnI have been rendered nonphosphorylatable by mutation to alanine.

Figure 1

Immunoaffinity purification of cTn complexes from mouse hearts. SDS–PAGE analysis of affinity purified cardiac troponin complex stained with Coomassie Blue. M, molecular weight markers, C, chicken cTn (obtained from Sigma-Aldrich), EX, extracted cardiac myofilament proteins; FLT, column flow through; W, wash; EL, elution of troponin complex.

Figure 2

High resolution MS analysis of intact cTnI purified from wild-type mouse hearts. Bottom panel: ESI/FTMS spectrum of cTnI showing it is mono- and bis-phosphorylated. N-terminally proteolytic degraded products of cTnI were also observed. Top panel: isotopically resolved molecular ions of un-, mono-, and bis-phosphorylated cTnI (M²⁹⁺) with highly accurate molecular weights measured. _pcTnI and _ppcTnI represent mono- (+79.97 Da, HPO₃), and bis-phosphorylated (+159.92 Da, 2 HPO₃) cTnI. +Na indicates cTnI ion with sodium adduct peaks. Circles represent the theoretical isotopic abundance distribution of the isotopomer peaks corresponding to the assigned mass. Dashed arrow indicates the expected position of tris-phosphorylated cTnI (_pppcTnI), which is not observed here. Calc’d, calculated most abundant molecular weight; Expt’l, experimental most abundant molecular weight.

Figure 3

Gas-phase separation (purification) of cTnI phosphorylated ions. Isolation of a single charge state (M²⁹⁺) of a mixture of un-, mono-, and bis-phosphorylated cTnI (A), monophosphorylated cTnI alone (B), and bis-phosphorylated cTnI alone (C). _pcTnI and _ppcTnI represent mono-, and bis-phosphorylated cTnI, respectively.

Figure 4

Localization of basal phosphorylation sites to Ser22/23 in wild-type mouse cTnI by ECD mass spectrometry. (I–IV) Representative spectra of doubly charged (2⁺) c₂₁–c₂₄ fragment ions, respectively, from ECD of a single charge state (M²⁹⁺) of a mixture of un-, mono-, and bis-phosphorylated cTnI (A), monophosphorylated cTnI alone (B), and bis-phosphorylated cTnI alone (C). _pc and _ppc represent mono- and bis-phosphorylated c fragmentation ions. The abbreviated amino acid sequences for ions c₂₁–c₂₄ were accordingly denoted on top of each panel. The positively identified isotopomer peak profiles of un-, mono-, and bis-phosphorylated c₂₁–c₂₄ (2⁺) ions were correspondingly expanded in each spectrum. Circles represent the theoretical isotopic abundance distribution of the isotopomer peaks corresponding to the assigned molecular weight. Calc’d, calculated most abundant molecular weight; Expt’l, experimental most abundant molecular weight.

Figure 5

MS/MS product map from the ECD spectra for assignments to mouse cTnI. Fragment assignments were made to the DNA-predicted sequence of mouse cTnI (UnitProtKB/Swiss-Prot P48787, TNNI3_mouse) with the removal of N-terminal methionine and acetylation at the new terminus. (A) ECD of a single charge state (M²⁹⁺) of a mixture of un-, mono-, and bis-phosphorylated cTnI; (B) ECD of one charge state (M²⁹⁺) of monophosphorylated cTnI alone; (C) ECD of a single charge state (M²⁹⁺) of bis-phosphorylated cTnI alone. Square, both un- and monophosphorylated fragment ions were observed. Star, un-, mono-, and bis-phosphorylated ions were observed. Single dot, only monophosphorylated fragment ions observed. Double dots, only bis-phosphorylated fragment ions were observed. Ser22 and Ser23 are highlighted in circles.

Figure 6

(A) High resolution MS analysis of cTnI affinity purified from cTnI-Ala₂ transgenic mouse hearts. No phosphorylated ions were detected, confirming Ser22 and Ser23 are the only basally phosphorylated sites in wild-type mouse cTnI. N-terminally proteolytic degraded products of cTnI were also observed. Inset, isotopically resolved molecular ions of unphosphorylated cTnI (M²⁹⁺) with highly accurate molecular weight measured. Dashed arrow indicates the expected position of monophosphorylated cTnI from cTnT-Ala2 transgenic mice (_pcTnI-Ala2), which is not observed here. +Na indicates cTnI ion with sodium adduct peaks. Circles represent the theoretical abundance isotopic distribution of the isotopomer peaks corresponding to the assigned mass. Calc’d, calculated most abundant molecular weight; Expt’l, experimental most abundant molecular weight. (B) SDS–PAGE analysis of myofilament proteins extracted from both wild-type and cTnI-Ala₂ transgenic mouse hearts stained with Pro-Q diamond. Only lower molecular weight regions are shown. Lane 1, WT, wild-type mouse; lane 2, Ala2, cTnI-Ala₂ transgenicmouse. cTnT, cardiac troponin T; Tm, tropomyosin; LC₂, myosin regulatory light chain 2.