Top-down Proteomics Reveals Concerted Reductions in Myofilament and Z-disc Protein Phosphorylation after Acute Myocardial Infarction*

Heart failure (HF) is a leading cause of morbidity and mortality worldwide and is most often precipitated by myocardial infarction. However, the molecular changes driving cardiac dysfunction immediately after myocardial infarction remain poorly understood. Myofilament proteins, responsible for cardiac contraction and relaxation, play critical roles in signal reception and transduction in HF. Post-translational modifications of myofilament proteins afford a mechanism for the beat-to-beat regulation of cardiac function. Thus it is of paramount importance to gain a comprehensive understanding of post-translational modifications of myofilament proteins involved in regulating early molecular events in the post-infarcted myocardium. We have developed a novel liquid chromatography–mass spectrometry-based top-down proteomics strategy to comprehensively assess the modifications of key cardiac proteins in the myofilament subproteome extracted from a minimal amount of myocardial tissue with high reproducibility and throughput. The entire procedure, including tissue homogenization, myofilament extraction, and on-line LC/MS, takes less than three hours. Notably, enabled by this novel top-down proteomics technology, we discovered a concerted significant reduction in the phosphorylation of three crucial cardiac proteins in acutely infarcted swine myocardium: cardiac troponin I and myosin regulatory light chain of the myofilaments and, unexpectedly, enigma homolog isoform 2 (ENH2) of the Z-disc. Furthermore, top-down MS allowed us to comprehensively sequence these proteins and pinpoint their phosphorylation sites. For the first time, we have characterized the sequence of ENH2 and identified it as a phosphoprotein. ENH2 is localized at the Z-disc, which has been increasingly recognized for its role as a nodal point in cardiac signaling. Thus our proteomics discovery opens up new avenues for the investigation of concerted signaling between myofilament and Z-disc in the early molecular events that contribute to cardiac dysfunction and progression to HF.


Experimental Procedures
Generation and functional characterization of swine AMI model. We induced AMI in swine via occlusion of the left anterior descending (LAD) coronary artery using an angioplasty balloon and a percutaneous femoral approach similar to that reported previously ( Fig. 1) (1). All experiments involving animals were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and using protocols approved by the University of Wisconsin Institutional Animal Care and Use Committee. Yorkshire swine (30-35 kg) from the University of Wisconsin-Madison colony were pre-medicated with a mixture of telazol (4-6 mg/kg IM), xylazine (2 mg/kg,IM), and propofol (2-8 mg/kg IV), intubated, anesthetized, and mechanically ventilated with isoflurane (1-5%). A right carotid artery cutdown was performed, a 6Fr sheath inserted, and a 5.0Fr VSL pressure-volume (PV) catheter (Scisense Inc., London, Ontario) advanced to the left ventricle (LV). A bolus of heparin [50 U/kg, Intravenous (IV)] was given at this time and 25 U/kg was administered each hour thereafter. The catheter was connected to the ADVantage System (Transonic Sciscence, Ithaca, NY) linked to recording and analysis software (Iworx 404, Iworx Systems Inc., Dover, USA). The catheter measures blood and parallel conductance in real-time eliminating the need for saline bolus. Blood resistivity was assumed to be constant in all animals (150 cm). Catheter positioning was optimized using fluoroscopy and by examining the PV trace. Trans-femoral arterial and venous access was obtained. A 23 mm diameter balloon (Edwards Lifesciences, Irvine, CA) was advanced to the inferior vena cava just cranial to the diaphragm. Baseline PV loops were obtained and the vena cava balloon was briefly inflated to alter loading conditions with the ventilator turned off. Next, a coronary balloon catheter was introduced into the proximal LAD just distal to the first diagonal arterial branch and inflated for 90 min (AMI group, n=6). Iodinated contrast was used to confirm complete cessation of coronary blood flow distal to the balloon. After 90 min, PV loop data was again collected with the catheter in place and the LAD still occluded. In control animals (CON; n=6), PV analysis was performed and then the animals were sacrificed, and tissue was harvested as detailed below. Oxygen saturation, ECG, end tidal CO 2 , and arterial blood pressure were continuously monitored and remained within normal ranges for the duration of the studies. After the final PV-loop was obtained, the animal was euthanized with saturated KCl and the heart was removed and sectioned. Sections were either rapidly frozen in liquid nitrogen or placed in 10% buffered formalin for histology. 90 min occlusion of the lower one-third of the LAD created an ischemic zone comprising roughly 25% of the LV, less than 5% of the right ventricle (RV), and 5-10% of the interventricular septum (IVS).
Protein identification. The raw MS/MS data were converted to mzXML files and deconvoluted with MS-Deconv (2), a combinatorial algorithm that determines the monoisotopic masses and charge for all of the fragment ions present in an MS/MS spectrum. Subsequently, to identify the proteins present in the sample, the files containing the monoisotopic mass, intensity, and charge for each of the product ions were searched against the pig database generated from NCBI (Sscrofa10.2, containing 24,476 protein sequences) using the MS-Align+ algorithm (3). Here, 10 ppm mass measurement accuracy was utilized as a cut-off for MS/MS fragment assignments.
The use of both CAD and ECD MS/MS spectra for various charge states of the same protein resulted in unique protein identification with significantly lower statistical P values and a higher number of assigned fragments correlating directly to a higher confidence level in protein identification. All protein identifications were validated manually.
Moreover, MS-Align+ is a fast algorithm for top-down protein identification based on spectral alignment that allows for the identification of sequence variations and PTMs as described previously (3,4). Because the target proteoforms were not included in the protein database, which contains only unmodified proteoforms, MS-Align+ reported a homologous unmodified proteoform for each deconvoluted top-down MS/MS spectrum. Compared with the homologous proteoforms, the target proteoforms contained sequence variations and/or PTMs.
Based on the proteoform-spectrum-match (PrSM) reported by MS-Align+, the first sequence modification (sequence variation or PTM) was identified and localized manually. Then the sequence variation or PTM was added to the homologous proteoform to generate a new proteoform for the identification of the second sequence variation or PTM. Similarly, spectral alignment and manual inspection were used to identify the second sequence variation or PTM.
This process was iterated until all sequence variations and PTMs were identified and localized.

Results
Identification of proteins from myofilament protein-enriched extracts. We separated over twenty proteins in myofilament protein-enriched extracts (Fig. 2b, Fig. S2). Although we were able to preliminarily identify key myofilament proteins such as cardiac troponin T (cTnT), cardiac troponin I (cTnI), tropomyosin (Tm), myosin essential light chain (MLC1), myosin regulatory light chain (MLC2), actin, and troponin C (TnC) based on their intact relative molecular weight (M r ), which closely matched the sequences available in the database for these proteins, a significant number of the proteins detected could not be identified solely based on accurate determination of their M r (Fig. S2, Table S2). Therefore, we collected the fractions and employed high-resolution FT-ICR MS/MS for protein identification. Several charge states of precursor ions per protein from each individual fraction were isolated and subsequently fragmented by collisionally activated dissociation (CAD) and electron capture dissociation (ECD). The product ions were then searched against the swine database (FASTA file) generated from NCBI with MS-Align+ algorithm(3) for intact protein identifications (Table S2).
These proteins were reported previously to be present in the myofilament subproteome (5) .

MS/MS allows for the comprehensive characterization of proteoforms and the localization of
PTMs and sequence variations. The comprehensive characterization of Tm from AMI myocardium is provided as an example (Fig. S9). We identified N-terminal acetylation, two amino acid polymorphisms (Pro38→Gln38 and Pro64→Leu64), and a phosphorylation site at Ser283, as reported previously (6). Besides the major proteoforms for Tm (-Tm, p -Tm, -Tm, p -Tm), four minor proteoforms with lower abundance (Tm-U1, Tm-U2, Tm-U3 and Tm-U4 in the spectrum, with mass differences of +312, +328, +378 and +393 Da from predominant α-Tm, respectively) were also detected (Fig. 4c). Due to the lack of significance between CON and AMI and the relative low abundance (Fig. 4e), these proteoforms were not investigated further.
Mapping phosphorylation sites in swine cTnI to Ser22/23. To identify the phosphorylation sites of swine cTnI, the precursor ions of p cTnI and pp cTnI were isolated and subsequently fragmented using ECD ( Fig. 5A and Fig. S10). In five combined ECD spectra of p cTnI, 91 c ions and 107 z· ions were detected. Note that c ions count from the N-terminus and z· ions count from the C-terminus (e.g., c 21 contains the first 21 amino acids counting from the N-terminus and z· 21 contains the first 21 amino acids beginning from the C-terminus). While no ions before c 21 were phosphorylated, all of the c ions after c 21 were detected in their phosphorylated forms (including c 22 ), thus confirming Ser22 as the sole site of p cTnI phosphorylation in CON and AMI myocardium (Fig. S10). MS/MS data matches best with the sequence of swine cTnI (UnitProtKB/Swiss-Prot, A5X5T5, TNNI3_pig) after considering deletion of the N-terminal Met and acetylation at the new N-terminus, as well as the single amino acid polymorphism (V116A) and phosphorylation at Ser22 (Fig. S10), as we reported previously (7). For pp cTnI, a total of 96 c ions and 105 z· ions were detected, which unambiguously localized the phosphorylation sites to Ser22/23 (Fig. 5a). Since a clean isolation of precursor pp cTnI ions was obtained before ECD fragmentation and no un-or mono-phosphorylated counter ions were detected between Ser23 and Arg27, we conclude that full phosphorylation occupancy occurs on Ser22 and Ser23 (Fig.   5a).

Mapping the phosphorylation site in ENH2.
To locate the phosphorylation site in swine ENH2, the precursor ion of swine p ENH2 was isolated and then fragmented by ECD. In five combined ECD spectra of mono-phosphorylated ENH2, 58 c ions and 71 z· ions were detected (Fig. 5c). Among all the fragment ions, no phosphorylated product c ions were detected from the N-terminus to c 103, which indicated that the phosphorylation site was not located before Cys103 (Fig. S14). On the other hand, all of the c ions after c 119 were phosphorylated, suggesting that the phosphorylation site is located in the region between Cys103 and Glu119 (Fig. S14-15). In addition, the fact that all of the z· ions after z· 118 were phosphorylated while none of the z ions prior to z• 115 were phosphorylated provides further evidence that the site of phosphorylation is located on one of the amino acid residues between Arg116 to Ser118 (Fig. S14-15). Since there is only one phosphorylatable residue (Ser118) in this region (RGS), we can confidently assign Ser118 as the sole site of phosphorylation in ENH2.
Thus, our top-down MS data unambiguously confirms that the proteoform with the M r of 25,905.42 is the monophosphorylated form of swine ENH2 that is significantly reduced in the AMI heart compared with CON hearts (Fig. 4d). Supplemental Tables   Table S1. Hemodynamic data from CON and AMI swine hearts. Values are means ± SEM. ESP, end systolic pressure; SV, stroke volume; EF%, percent ejection fraction; PRSW, preload recruitable stroke work. *p < 0.05 using paired student t-test. CON, control; AMI, acute myocardial infarction.    Table S2. (A) Peak #7 was identified as mitochondrial ATP synthase subunit e, with N-terminal Met loss and a potential modification (+12.04 Da) in the C-terminal 4 residues (STLK); (B) Peak#14 was identified as histone H2B type 2-E-like with N-terminal Met loss; (C) Peak #15 was identified as cytochrome c oxidase subunit 5A, mitochondrial-like isoform 1 with an unidentified N-terminal modification; and (D) like isoform 1 with N-terminal Met loss and a potential modification on one of the internal residues (NFKLLSH); and (E) Peak # 9 was identified as PDZ-LIM domain protein 5 isoform 2, also known as enigma homolog isoform 2 (ENH2). MS-Align+ is a fast algorithm for top-down protein identification based on spectral alignment that enables searches for PTMs. PrSM ID is the identification number of a protein-spectrum-match reported by MS-Align+. The E-value of a PrSM describes the number of hits one can -expect‖ to see by chance when searching the e d spectrum against a protein database with the same size to the target database. The P-value of a PrSM stands for the probability that the match between the spectrum and a random protein has a similarity score no less than that the score of the PrSM.

Figure S4. High reproducibility of the MS method evaluated by technical replicates.
Representative MS data are shown here. (A) Overlay of low-resolution MS spectra for two injection replicates from the same TFA extraction. (B) Three extraction replicates (replicates of different TFA extractions from the same tissue) were aligned vertically (note the baseline of the top two MS spectra were elevated intentionally for better visualization). Proteins were extracted from CON myocardium. Mono-and bis-phosphorylated proteins are labeled with a " p " and " pp ", respectively. Stars indicate non-covalent phosphoric acid adducts.

Figure S5. Rapid MS profiling of key myofilament and associated proteins by on-line LC/MS analysis.
Comparison of MS spectra of cTnT, cTnI, Tm, MLC1, MLC2, actin, and ENH2 from CON (A, C, E) and AMI (B, D, F) myocardium, respectively. Similar MS profiles were observed among biological replicates showcasing the high reproducibility of our method. Significant differences were detected for cTnI, MLC2, and ENH2 phosphorylation in CON and AMI hearts. Mono-and bis-phosphorylated proteins are labeled with a " p " and " pp ", respectively. Stars indicate non-covalent phosphoric acid adducts.  , and ENH2 (D) extracted from CON and AMI swine hearts. Mono-and bis-phosphorylated proteins are labeled with a " p " and " pp ", respectively. Circles, the theoretical isotopic abundance distribution of the isotopomer peaks corresponding to the assigned mass. Exp, observed most abundant molecular weight. Figure S8. Quantification of myofilament and associated protein phosphorylation based on high-resolution MS analysis. Quantification of the total phosphorylation level of cTnI, MLC2, α-Tm, β-Tm, and ENH2 extracted from CON and AMI hearts. All measurements are based on data from three animals per group. Each biological replicate was performed in two technical replicates. The bar graph data are represented as the mean peak area ± S.E.M. Box, median and interquartile range (25%, 75%); Whiskers, minimum and maximum values. *p<0.05. ** p<0.001. TPM1_PIG) with acetylation of the first amino acid at the N-terminus (+42.01 Da) and two amino acid polymorphisms, P38Q (-28.04 Da) and P64L (16.03 Da). A single phosphorylation site was localized to Ser283 similarly as reported previously (6).

Figure S10. Comprehensive characterization of p cTnI by high-resolution ECD MS/MS.
Fragmentation maps of p cTnI based on 5 ECD spectra. The phosphorylation site is labeled with -p‖. -Ac-‖ denotes acetylation. Both MS and MS/MS data matches best with the sequence of swine cTnI (UnitProtKB/Swiss-Prot, A5X5T5, TNNI3_pig) after considering the deletion of Nterminal Met and acetylation at new N-terminus as well as the single amino acid polymorphism (V116A) and a phosphorylation site at Ser22 as we reported previously(7).   Figure S13. Sequence alignment of ENH2 from different species. The region containing the potential amino acid substitutions between human, pig, and mouse based on the MS/MS data are marked in purple. Asterisks denote conserved amino acids. Human (Q96HC4-2) and mouse (Q8CI51-2) ENH2 sequences are retrieved from the Swiss-Prot database. The swine ENH2 sequence is predicted based on the genomic sequence in pig database (Sscrofa10.2) from NCBI. ENH2 is also known as PDZ-LIM domain protein 5 isoform 2-referred to as PDLI5-2_Pig in the figure.
Figure S14. MS/MS for sequence characterization of mono-phosphorylated ENH2 ( p ENH2) and localization of the phosphorylation site. (A) Fragmentation assignments are made to the predicted swine ENH2 sequence (PDLI5-2) from NCBI pig database (Sscrofa10.2) without any modifications. Note that only 50 z· ions matched the unmodified ENH2 sequence. (B) Fragment assignments are made to predicted swine ENH2 sequence with deletion of the N-terminal Met and acetylation of the new N-terminus. 22 c ions were assigned in addition to 50 z· ions. (C) Fragment assignments are made based on the predicted swine ENH2 sequence with acelylation of the new N-teminus and one amino acid substitution (Ser72→Cys72). 30 c ions were assigned in addition to 50 z· ions. (D) Fragment assignments are made to the predicted ENH2 sequence with acelylation of the new N-teminus, one amino acid substitution and localization of the phosphorylation site at Ser118. Note that a total of 58 c ions and 71 z· ions were assigned. The amino acid substitution is highlighted in orange andp ‖ represents phosphorylation site at Ser118. Thus we fully characterized the sequence of p ENH2 with deletion of N-terminal Met, acetylation of the new N-terminus, an amino acid substitution at Ser72Cys, and phosphorylation site at Ser118. Phosphorylation sites are highlighted by circles. Amino acid polymorphism (Ser72Cys) is highlighted in heptagon.

Figure S15. MS/MS mapping of the phosphorylation site in swine ENH2. (A-F)
Representative MS/MS spectra of c and z· ions from ECD spectra of mono-phosphorylated ENH2. The site of mono-phosphorylation was localized to Ser118. The assignment of fragment ions was made based on the predicted sequence of swine ENH2 (PDLI5-2) from NCBI pig database (Sscrofa10.2) with deletion of the N-terminal Met, acetylation of the new N-terminus, and one amino acid substitution (Ser72→Cys72). Figure S17. Comparable charge state distributions of ENH2 despite different phosphorylation levels between CON and AMI. Main spectra: Representative full charge state profiles of ENH2 from on-line LC/MS analysis of myofilament subproteome extracted from CON and AMI hearts respectively. Insets, zoom-in spectra of a single charge state (M 37+ ) of ENH2 showing the difference in phosphorylation levels of ENH2 between AMI and CON.