Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state

Abstract

Cardiac myosin binding protein C (cMyBP-C), bound to the sarcomere's myosin thick filament, plays an important role in the regulation of muscle contraction. cMyBP-C is a large multidomain protein that interacts with myosin, titin, and possibly actin. Mutations in cMyBP-C are the most common known cause of heritable hypertrophic cardiomypathies. Phosphorylation of cMyBP-C plays an essential role in the normal cardiac function. cMyBP-C (142 kDa) has 81 serine and 73 threonine residues presenting a major challenge for unequivocal identification of specific phosphorylation sites. Top-down mass spectrometry, which directly analyzes intact proteins, is a powerful technique to universally observe and quantify protein posttranslational modifications without a priori knowledge. Here, we have extended top-down electron capture dissociation mass spectrometry to comprehensively characterize mouse cMyBP-C expressed in baculovirus. We have unambiguously identified all of the phosphorylation sites in the truncated (28-115 kDa) and full-length forms of cMyBP-C (142 kDa) and characterized the sequential phosphorylations, using a combination of top-down and middle-down (limited proteolysis) MS approach, which ensures full sequence coverage. Unit mass resolution and high mass accuracy (<5 ppm) have been achieved for a 115-kDa protein (the largest protein isotopically resolved to date). Remarkably, we discovered that truncations in recombinant proteins, even a seemingly minor one, can dramatically alter its phosphorylation state, which is significant because truncated recombinant proteins are routinely substituted for their full-length forms in crystal structure and functional studies. Our study provides direct evidence of alterations in the posttranslational state between the truncated and full-length recombinant proteins, which can lead to variations in structure and function.

Fig. 1.

Baculovirus expressed cMyBP-C constructs. (A) Schematic representation of the 11 domains (8 IgI and 3 Fn) of cMyBP-C, full-length, and truncated forms compared with fast/slow skeletal MyBP-C. (B) SDS/PAGE analysis of full-length and truncated cMyBP-C stained with Coomassie Blue. Molecular weight standards (× 10⁻³) are indicated on the left.

Fig. 2.

High resolution ESI/FTMS analysis of cMyBP-C C0-C1 expressed in baculovirus. (Upper) ESI/FTMS spectrum of C0-C1 intact protein ions (M³²⁺), suggesting C0-C1 is acetylated and prevailingly unphosphorylated. (Lower) Fragmentation map from 1 ECD and 1 CAD spectra of C0-C1 matched with assignments to the DNA-predicted sequence of C0-C1 with an N-terminal acetylation.

Fig. 3.

High resolution ESI/FTMS analysis of C0-C4 expressed in baculovirus. (Upper) ESI/FTMS spectrum of C0-C4 intact protein ions (M⁹⁰⁺), suggesting C0-C4 is acetylated and mono-, bis-, and tris-phosphorylated. Un-P, mono-P, bis-P, and tris-P stand for the un-, mono-, bis-, and tris-phosphorylated C0-C4. (Lower) Fragmentation map from 3 ECD and 4 CAD spectra of C0-C4 matched with assignments to the DNA-predicted sequence of C0-C4 with an N-terminal acetylation.

Fig. 4.

High resolution ESI/FTMS analysis of ΔC0-C1 expressed in baculovirus. (Upper) ESI/FTMS spectrum of ΔC0-C1 intact protein ions (M¹⁰¹⁺), suggesting ΔC0-C1 is acetylated and monophosphorylated. (Lower) Fragmentation map from 3 ECD and 4 CAD spectra of ΔC0-C1 matched with assignments to the DNA-predicted sequence of ΔC0-C1 with an N-terminal acetylation.

Fig. 5.

Comparison of ESI/MS spectra of full-length and truncated cMyBP-C. (A) High resolution ESI/FTMS spectrum of full-length cMyBP-C expressed in baculovirus. (B–D) Low resolution ESI/LTQMS spectrum of full-length cMyBP-C, C0-C10 (142 kDa) and truncated cMyBP-C, ΔC0-C1 (115 kDa), and C0-C4 (71 kDa). Un-P, mono-P, bis-P, and tris-P represent the un-, mono-, bis-, and tris-phosphorylated molecular ions, respectively.

Fig. 6.

Identification of phosphorylation sites from limited Asp-N proteolysis of full-length cMyBP-C. (A) ECD spectrum of monophosphorylated D[274–315]R (_pD[274–315]R) localizing Ser-292 as the phosphorylation site. (B) ECD spectrum of bisphosphorylated D[274–315]R (_ppD[274–315]R) localizing Ser-292 and Ser-312 as the phosphorylation sites. The identified phosphorylation sites and the phosphorylated c/z^• fragmentation ions are labeled with a “p.”