Human myocardial protein pattern reveals cardiac diseases

Abstract

Proteomic profiles of myocardial tissue in two different etiologies of heart failure were investigated using high performance liquid chromatography (HPLC)/Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Right atrial appendages from 10 patients with hemodynamically significant isolated aortic valve disease and from 10 patients with isolated symptomatic coronary heart disease were collected during elective cardiac surgery. As presented in an earlier study by our group (Baykut et al., 2006), both disease forms showed clearly different pattern distribution characteristics. Interesting enough, the classification patterns could be used for correctly sorting unknown test samples in their correct categories. However, in order to fully exploit and also validate these findings there is a definite need for unambiguous identification of the differences between different etiologies at molecular level. In this study, samples representative for the aortic valve disease and coronary heart disease were prepared, tryptically digested, and analyzed using an FT-ICR MS that allowed collision-induced dissociation (CID) of selected classifier masses. By using the fragment spectra, proteins were identified by database searches. For comparison and further validation, classifier masses were also fragmented and analyzed using HPLC-/Matrix-assisted laser desorption ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) mass spectrometry. Desmin and lumican precursor were examples of proteins found in aortic samples at higher abundances than in coronary samples. Similarly, adenylate kinase isoenzyme was found in coronary samples at a higher abundance. The described methodology could also be feasible in search for specific biomarkers in plasma or serum for diagnostic purposes.

Figure 1

Flowchart for the path of the sample preparation and measurements with 9.4 Tesla FT-ICR MS. The measurement of the samples with two different instrument had the reason that the samples were measured with a classical FT-ICR instrument without external MS/MS capability. After the differential mass spectrometric runs followed by the pattern comparison, a quadrupole/hexapole FT-ICR instrument with external MS/MS capability was available. The fragmentation studies for the protein identification were performed with this latter instrument.

Figure 2

Non-scaled schematic view of an LC-FT-ICR MS system with a quadrupole mass selector and a hexapole collision cell. In the electrospray ion source the formed ions are captured after they pass the electrospray capillary in an ion funnel, which increases the sensitivity of the system by roughly up to an order of magnitude. Ions are transferred through two ion funnels into a hexapole ion guide, where they can also be trapped. Ions are selected in the quadrupole mass selector and can undergo collision induced dissociation in the hexapole collision chamber which is at a relatively high pressure. The ICR cell is in the magnetic center of a 9.4T superconducting magnet. The vacuum system, not shown in the figure, consists of pumping stages down to the range of 10⁻¹⁰ mbar in the ultra high vacuum chamber of the ICR cell. The numbers shown are approximate pressures in different pumping stages.

Figure 3

Non-scaled schematic view of a MALDI-TOF/TOF mass spectrometer.

Figure 4

Examples of data (m/z versus number of accumulated scans (corresponds to LC retention time), 10 seconds each) obtained from HPLC-FT-ICR mass spectrometry of aortic (a) and coronary (b) samples. The light spots in the diagrams correspond to individual mass spectral peaks. In the original diagrams the peak intensities are color coded for easy recognition. (Figure modified from reference [9] with kind permission from the publisher).

Figure 5

Pattern for the classification of coronary (circular symbols) versus AVD disease (square symbols) samples. Each point represent an individual sample where filled symbols (● and ■) represent supervised classified training samples while open symbols (○ and □) represent unsupervised classified test samples. All samples with exception of two were unambiguously correctly classified. (Figure modified from reference [9] with kind permission from the publisher).

Figure 6

MS/MS spectra of the classifier peptide FLEQQNAALAAEVNR from the sample of an aortic patient. The protein is identified as Desmin upon database search. Spectrum (a) is obtained by LC-ESI-FTMS/MS, spectrum (b) by LC-MALDI-TOF/TOF.

Figure 7

MS/MS spectra of the classifier peptide GQLVPLETVLDMLR from the sample of an aortic patient. The protein is identified as Adenylate Kinase upon database search. Spectrum (a) is obtained by LC-ESI-FTMS/MS, spectrum (b) by LC-MALDI-TOF/TOF.

Figure 8

MS/MS spectra of the classifier peptide GSSFQTVSALHR from the sample of an aortic patient. The protein is identified as Myosin Heavy Chain Beta Isoform upon database search. Spectrum (a) is obtained by LC-ESI-FTMS/MS, spectrum (b) by LC-MALDI-TOF/TOF.

Figure 9

MS/MS spectra of the classifier peptide IWHHTFYNELR from the sample of an aortic patient. The protein is identified as Beta Actin upon database search. Spectrum (a) is obtained by LC-ESI-FTMS/MS, spectrum (b) by LC-MALDI-TOF/TOF.

Figure 10

Proteomic profiles of myocardial tissue in two different etiologies of heart failure were investigated using right atrial appendages samples representative for the aortic valve disease and coronary heart disease using a quadrupole/hexapole FT-ICR MS that allowed collision induced dissociation (CID) of selected classifier masses. For comparison and further validation, classifier masses were also fragmented and analyzed using HPLC/Matrix assisted laser desorption ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) mass spectrometry.