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.
Understanding the differences of proteomic profiles has a crucial importance for gaining insight into molecular mechanisms of disease. Although the molecular origin of the cardiac dysfunction is still largely unknown in the majority of heart diseases [
The objective of this study is to evaluate selected individual myocardial samples which are representative for aortic valve disease (AVD) and coronary heart disease (CHD), respectively. As presented in our recent study [
As a general overview for the method used, a flow chart is shown in Figure
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.
In 20 patients undergoing cardiac surgery with extracorporeal circulation, right atrial appendages were subject to removal and discarding for venous cannulation that were collected intraoperatively, after approval by the local ethical committee. The group of patients consisted of 10 individuals with hemodynamically significant, isolated AVD and 10 with isolated symptomatic CHD. Patient selection was made in such a way that all patients with CHD selected for this study were “healthy” in terms of the AVD and all selected AVD patients were “healthy” in terms of the coronary disease. The median age was 62 years (45–81) in the AVD group, and 66 years (37–83) in the CHD group. All antithrombotic agents were suspended 10–14 days prior to surgery. In all studied cases, patients stopped taking aspirin one week before the operation. Cardiac samples (each of them in the range of 1 cm3) were immediately washed in Krebs-Henseleit solution (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.25 mM CaCl2, 1.2 mM KH2PO4, 25 mM NaHCO3, 11 mM glucose) and fixed on wax plates at room temperature. After separation from the epicardium, the trabecular tissue was shock-frozen in liquid nitrogen and stored at −75°C.
Sample selection and preparation of the tissue specimens have been described in detail in our earlier study [
The total amount of each digested sample was dissolved in 250
Mass spectra were acquired with an APEX Qe FT-ICR (Bruker Daltonics Inc., Billerica, MA, USA) equipped with a 9.4 Tesla superconducting magnet (Bruker BioSpin, Wissembourg, France). A schematic description of the LC-FT-ICR MS system with a quadrupole mass selector and a hexapole collision cell is shown in Figure
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.
The mass spectra were processed using the protein analysis software Biotools 3.0 (Bruker Daltonics, Billerica, MA, USA) for database searches and results interpretation. The MASCOT search engine (Matrix Science, London, UK) [
The capillary high-performance liquid chromatography (HPLC) separations were performed using an Agilent 1100 Cap-LC system equipped with a 15 cm × 180
The MALDI-TOF-MS instrument used was an Ultraflex II TOF/TOF (Bruker Daltonik GmbH, Bremen, Germany) [
Non-scaled schematic view of a MALDI-TOF/TOF mass spectrometer.
Despite a 90 min separation of the peptides in the HPLC and a spread of fractions onto 384 spots, the acquired MS spectra were still very complex. On one side, complexity increased suppression effects during desorption/ionization, on the other side, selection of individual peptides for MS/MS often was ambiguous. In order to simplify MS spectra and avoid mixing of fragments from different precursor ions prior to the LC separation, selected samples were prefractionated using Magnetic Beads based on Weak Cation Exchange Chromatography (Profiling Kit 100 MB-WCX, Bruker Daltonik GmbH, Bremen, Germany). The freeze-dried samples were dissolved in 10
The target of choice for the LC-MALDI approach was a prespotted Anchor Chip target (Bruker Daltonik GmbH, Bremen, Germany). Within a 90 min LC run, all 384 sample spots on the target were prepared. After evaporation of the solvent, the target was washed by dipping it for a few seconds into a solution of 10 mM ammonium citrate/0.1% TFA. The sample plate was then introduced into the Ultraflex II MALDI-TOF mass spectrometer, and mass spectra were recorded from each prepared sample spot. The calibration used was an external near-neighbor calibration by means of additional 96 prespotted calibration spots. The sample used for calibration was a mixture of peptides covering the mass range from 700 Da–3500 Da already prepared onto the disposable target. The acquisition process was controlled by the WARP-LC software (Bruker Daltonik GmbH, Bremen, Germany) and a compound list was created. From these peptide masses, scored according to their signal-to-noise and the complexity of the MS spectrum, the candidates of possible biomarkers were selected manually and their MS/MS spectrum was recorded.
The spectra were processed by means of Flex Analysis and the peak lists were sent to BioTools for the database search using Mascot (Matrix Science, London, UK, [
The pattern recognition and classification strategy have been described in detail by Ramström et al. [
Examples of two-dimensional data obtained from HPLC-FT-ICR mass spectrometry are shown in Figure
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 [
As described in detail in our recent publication [
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 (
Using the digested proteins from chosen samples from patients with CHD as well as with AVD, selected classifier masses were fragmented by collision-induced dissociation in the hexapole collision chamber of the FT-ICR system. Fragmentation spectra of these peptides are used for the identification of the proteins by database search (MASCOT search, [
Proteins identified as classifiers in coronary samples by LC-FT-ICR MS/MS. On-line measurements with electrospray ionization. Proteins identified as classifiers in aortic samples by LC-FT-ICR MS/MS. On-line measurements with electrospray ionization.
FT-ICR MS/MS database search identification of classifier proteins from CHD patients
Protein ID in coronary samples | Peptide sequence | Sequence tag | Mascot score | |
---|---|---|---|---|
(P13533) Myosin heavy chain, cardiac muscle alpha isoform (MyHC-alpha) MYH6_HUMAN | KLAEKDEEMEQAK | 1577–1589 | 774.884 (2+), 516.925 (3+) | 65 (92), 28 |
NLQEEISDLTEQLGEGGKNVHELEKVR | 1506–1532 | 766.896 (4+) | 113 | |
(P12883) Myosin heavy chain, cardiac muscle beta isoform (MyHC-beta) MYH7_HUMAN | KLAEKDEEMEQAK | 1575–1587 | 774.884 (2+), 516.925 (3+) | 65, 28 |
(P00568) Adenylate kinase isoenzyme 1 (EC 2.7.4.3) (ATP-AMP transphosphorylase) (AK1) (Myokinase) KAD1_HUMAN | GQLVPLETVLDMLR | 64–77 | 792.447 (2+) | 105 |
(P45379) Troponin T, cardiac muscle (TnTc) (Cardiac muscle troponin T) (cTnT) TNNT2_HUMAN | VLAIDHLNEDQLR | 227–239 | 768.415 (2+) | 98 (86) |
(P62736) Actin, aortic smooth muscle (Alpha-actin-2) ACTA_HUMAN | MQKEITALAPSTMK | 315–328 | 774.912 (2+) | 18 |
FT-ICR MS/MS database search identification of classifier proteins in AVD patients
Protein ID in aortic samples | Peptide sequence | Sequence tag | Mascot score | |
---|---|---|---|---|
(P45379) Troponin T, cardiac muscle (TnTc) (Cardiac muscle troponin T) (cTnT) TNNT2_HUMAN | DLNELQALIEAHFENR | 107–122 | 956.482 (2+) | 93 |
(P17661) Desmin DESM_HUMAN | FLEQQNAALAAEVNR | 127–141 | 837.426 (2+) | 90 |
(P02144) Myoglobin MYG_HUMAN | HPGDFGADAQGAMNK | 119–133 | 505.894 (3+) | 71 (20) |
(P60709) Actin, cytoplasmic 1 (Beta-actin) ACTB_HUMAN or | IWHHTFYNELR | 85–95 | 505.922 (3+) | 70 (53) (55) |
(P68032) Actin, alpha cardiac (Alpha-cardiac actin) ACTC_HUMAN | IWHHTFYNELR | 87–97 | 758.379 (2+), 505.922 (3+) | 53, 61 |
(P51884) Lumican precursor (Keratan sulfate proteoglycan lumican) (KSPG lumican) LUM_HUMAN | ILGPLSYSK | 297–305 | 489.288 (2+) | 29 |
(P12111) Collagen alpha-3 (VI) chain precursor CO6A3_HUMAN | VAVVQYSDR | 1067–1075 | 518.776 (2+) | 59 |
(P12883) Myosin heavy chain, cardiac muscle beta isoform (MyHC-beta) MYH7_HUMAN | RKLEGDLK (also in MYH6_Human) | 1053–1060 | 479.788 (2+) | 21 |
AQLEFNQIK | 1561–1569 | 545.799 (2+) | 25 | |
GSSFQTVSALHR | 641–652 | 645.334 (2+) | 25 (35) (62) | |
(P13533) Myosin heavy chain, cardiac muscle alpha isoform (MyHC-alpha) MYH6_HUMAN | RKLEGDLK | 1055–1062 | 479.788 (2+) | 21 |
AQLEFNQIK | 1563–1571 | 545.799 (2+) | 25 (26) | |
GSSFQTVSALHR | 643–654 | 645.334 (2+), 430.559 (3+) | 25 (17), 47 | |
GKLSYTQQMEDLKR | 1306–1319 | 566.295 (3+) | 37 | |
(P09493) Tropomyosin 1 alpha chain (Alpha-tropomyosin) TPM1_HUMAN | MEIQEIQLK | 141–149 | 566.309 (2+) | 36 |
(P02768) Serum albumin precursor ALBU_HUMAN | KYLYEIAR | 161–168 | 528.299 (2+) | 29 |
(P02511) Alpha crystallin B chain (Alpha(B)-crystallin) (Rosenthal fiber component) (Heat-shock) pro-CRYAB_HUMAN | HFSPEELK | 83–90 | 493.751 (2+) | 28 |
(P09669) Cytochrome c oxidase polypeptide VIc precursor (EC 1.9.3.1) COX6C_HUMAN | KAGIFQSVK | 67–75 | 489.293 (2+) | 28 |
(P12235) ADP/ATP translocase 1 (Adenine nucleotide translocator 1) (ANT 1) ADP, ATP carrier protein (ADT1_HUMAN) | TAVAPIER | 23–30 | 856.487 (1+) | 24 (22) |
(P35555) Fibrillin-1 precursor FBN1_HUMAN | TICIETIK | 843–850 | 489.271 (2+) | 23 |
(P02768) Serum albumin precursor ALBU_HUMAN | KYLYEIAR | 161–168 | 528.298 (2+) | 22 |
(P19429) Troponin I, cardiac muscle (Cardiac troponin I) TNNI3_HUMAN | AKESLDLR | 162–169 | 466.264 (2+) | 19 |
(P06576) ATP synthase beta chain, mitochondrial precursor (EC 3.6.3.14) ATPB_HUMAN | FLSQPFQVAEVFTGHMGK | 463–480 | 675.008 (3+) | 16 |
Tables
Proteins identified as classifiers in coronary samples by LC-MALDI-TOF/TOF mass spectrometry. Samples separated by LC deposited on plates, which are then analyzed by MALDI TOF/TOF MS. Proteins identified as classifiers in aortic samples by LC-MALDI-TOF/TOF mass spectrometry. Samples separated by LC deposited on plates, which are then analyzed by MALDI TOF/TOF MS.
HPLC/MALDI TOF/TOF database search identification of classifier proteins in CHD patients
Protein ID of coronary samples | Peptide sequence | Sequence tag | Mascot score | Protein summary score | |
---|---|---|---|---|---|
Collagen HSCOLL NID: | GYPGNIGPVGAAGAPGPHGPVGPAGK |
327–352 | 2284.147 |
185 (91) | 88 |
(P12883) Myosin heavy chain, cardiac muscle beta isoform (MyHC-beta) MYH7_HUMAN | VIQYFAVIAAIGDR | 191–204 | 1535.858. | 72 | 88 (41.20) |
(Q05639) Elongation factor 1-alpha 2 (EF-1-alpha-2) | VETGILRPGMVVTFAPVNITTEVK | 267–280 | 2571.421 | 55 | 74.50 |
Hypothetical protein DKFZp686P07163. -Homo sapiens (Human). Q5HYB7_HUMAN | SSSLLIPPLETALANFSSGPEGGVMQPVR | 19–47 | 2954.529 | 80 (66) | 105 (94.90) |
AX885183 NID: | LFDQFFGEHLLESDLFPTSTSLSPFYLRPPSFLR | 23–56 | 4004.027 | 106 (34) | 133 (56.90) |
(P02511) Alpha crystallin B chain (Alpha(B)-crystallin) | LFDQFFGEHLLESDLFPTSTSL | 23–44 | 2543.234 | 128 | 131 |
Crystallin, alpha B (Homo sapiens) gi∣13937813 | LFDQFFGEHLLESDLFPTSTSL | 23–44 | 2543.234 | 110 | 111 |
Crystallin, alpha B (Homo sapiens) gi∣4503057 | LFDQFFGEHLLESDLFPTSTSL | 23–44 | 2543.234 | 95 | 95.3 |
Adenylate kinase (EC 2.7.4.3) 1-human (tentative sequence) KIHUA or | GQLVPLETVLDMLR | 64–77 | 1583.882 | 64 (49) | 86.10 (69.80) |
(MLRA_HUMAN) Myosin regulatory light chain 2, atrial isoform (Myosin light chain 2a) (MLC-2a) (MLC2a) (Myosin regulatory light chain 7) Myosin regulatory light Q01449 | QLLLTQADKFSPAEVEQMFALTPMDLAGNIDYK | 129–161 | 3697.849 | 106 | 131 |
(P45379) Troponin T, cardiac muscle | VLAIDHLNEDQLR | 227–239 | 1535.81 | 58 | 85 |
HPLC/MALDI TOF/TOF database search identification of classifier proteins in AVD patients
Protein ID of aortic samples | Peptide sequence | Sequence tag | Mascot score | Protein summary score | |
---|---|---|---|---|---|
(P17661) Desmin DESM_HUMAN | FLEQQNAALAAEVNR | 127–141 | 1673.860 | 115 | 133.00 |
mutant desmin (Homo sapiens) gi∣21358854 | FLEQQNAALAAEVNR | 128–142 | 1673.860 | 99 (65) | 117 (84.30) |
(P60709) Actin, cytoplasmic 1 (Beta-actin) ACTB_HUMAN or | IWHHTFYNELR | 85–95 | 1515.749 | 82 | 100 |
(P63261) Actin, cytoplasmic 2 (Gamma-actin) ACTG_HUMAN or | IWHHTFYNELR | 85–95 | |||
(P68133) Actin, alpha skeletal muscle (Alpha-actin 1) ACTS_HUMAN or | IWHHTFYNELR | 85–95 | |||
(P68032) Actin, alpha cardiac (Alpha-cardiac actin) ACTC_HUMAN | IWHHTFYNELR | 87–97 | |||
(P12883) Myosin heavy chain, cardiac muscle beta isoform (MyHC-beta) MYH7_HUMAN or | GSSFQTVSALHR | 641–652 | 1289.660 | 79 | 87.30 |
(P13533) Myosin heavy chain, cardiac muscle alpha isoform (MyHC-alpha) MYH6_HUMAN | GSSFQTVSALHR | 643–654 | 1289.660 | 79 | 85.70 |
MSTP161 (Homo sapiens) gi∣33338222 | SFPNLAFIR | 108–116 | 1064.589 | 74 | 97.40 |
Myosin light chain 2a (Homo sapiens) gi∣10864037 | SLCYIITHGDEKEE 3: Carbamidomethyl (C) | 162–175 | 1693.774 | 66 | 122 |
actin-like protein (Homo sapiens) gi∣62421180 | IWHHTFYNELR | 2–12 | 1515.749 | 64 | 88.40 |
Myosin heavy chain alpha subunit gi∣386971 | AQLEFNQIK | 13–21 | 1090.589 | 60 | 81 |
alpha-1 type III collagen gi∣180413 or | GDKGETGER | 7–15 | 948.438 | 57 | 87 |
unnamed protein product (Homo sapiens) gi∣1340174 or | 28–36 | 80.1 | |||
alpha1 (III) collagen (Homo sapiens) gi∣30054 or | 141–153 | 74.50 | |||
alpha-1 (III) collagen (Homo sapiens) gi∣930045 or | 945–953 | 71.40 | |||
III preprocollagen alpha 1 chain (Homo sapiens) gi∣16197601 | 1092–1100 | 70.10 | |||
(P12235) ADP, ATP carrier protein, heart/skeletal muscle isoform T1 (ADP/ATP translocase 1) ADT1_HUMAN or | TAVAPIER | 23–30 | 856.489 | 49 | 65.10 |
(P05141) ADP, ATP carrier protein, fibroblast isoform (ADP/ATP translocase 2) ADT2_HUMAN or | TAVAPIER | 23–30 | 856.489 | 49 | 65.10 |
(P12236) ADP, ATP carrier protein, liver isoform T2 (ADP/ATP translocase 3) ADT3_HUMAN | TAVAPIER | 23–30 | 856.489 | 49 | 65.10 |
Cytochrome c oxidase subunit Va, (COX5A protein). -Homo sapiens (Human). Q8TB65_HUMAN | RLNDFASTVR | 98–107 | 1178.628 | 49 | 70.40 |
(P12883) Myosin heavy chain, cardiac muscle beta isoform (MyHC-beta) MYH7_HUMAN or | ILYGDFR | 713–719 | 883.467 | 46 | 56.20 |
(P13533) Myosin heavy chain, cardiac muscle alpha isoform (MyHC-alpha) MYH6_HUMAN | ILYGDFR | 715–721 | 883.467 | 46 | 56.20 |
(P12883) Myosin heavy chain, cardiac muscle beta isoform (MyHC-beta) MYH7_HUMAN or | AVVEQTER | 1689–1697 | 931.484 | 38 | 49.20 |
unnamed protein product (Homo sapiens) gi∣34533821 | FLLVGQTMSTLLDEDLTK | 495–512 | 2024.062 | 29 | 47.90 |
Myosin, heavy polypeptide 7, cardiac muscle, beta variant (Homo sapiens) gi∣62088996 or | AGLLGLLEEMRDER 10: Oxidation (M) | 406–419 | 1617.826 | 29 | 42 |
(O14958) Calsequestrin, cardiac muscle isoform precursor (Calsequestrin 2) CASQ2_HUMAN | EHQRPTLR | 243–250 | 1036.565 | 24 | 41.60 |
alpha integrin interacting protein 63 (Homo sapiens) gi∣4468915 | ESVSSFVR | 27–35 | 910.463 | 25 | 39.60 |
Myosin, heavy polypeptide 7, cardiac muscle, beta variant (Homo sapiens) gi∣62088996 | GSSFQTVSALHR | 271–291 | 1289.660 | 47 | 63.60 |
Examples from MS/MS results acquired with FT-ICR and MALDI-TOF mass spectrometry as comparative cases are shown in Figures
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.
Aortic Sample LC-ESI-FTMS/MS, Desmin DESM_HUMAN
Aortic Sample LC-MALDI-TOF/TOF, Desmin DESM_HUMAN
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.
Coronary Sample LC-ESI-FTMS/MS, Adenylate Kinase KAD1_HUMAN
Coronary Sample LC-MALDI-TOF/TOF, Adenylate Kinase KAD1_HUMAN
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.
Aortic Sample LC-ESI-FTMS/MS, Myosin Heavy Chain Beta Isoform MYH7_HUMAN
Aortic Sample LC-MALDI-TOF/TOF, Myosin Heavy Chain Beta Isoform MYH7_HUMAN
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.
Aortic Sample LC-ESI-FTMS/MS, Beta Actin ACTB_HUMAN
Aortic Sample LC-MALDI-TOF/TOF, Beta Actin ACTB_HUMAN
In this work, the sample collection was made in such a way that all patients with CHD selected for this study were free from AVD, and all selected AVD patients were free from CHD. All comparisons presented in this work can, therefore, virtually be considered as to be “diseased” versus “healthy” case against each other. Differences in the mass chromatograms are determined as classifier masses. Thus, proteins identified from classifiers by this
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.
Some of the identified proteins appear both in the list of CHD samples as well as in the list of the AVD samples. As an example, myosin heavy chain alpha and beta isoforms have been found both in CHD and AVD cases (Tables
Classifier peptides in aortic and coronary diseases identifying myosin heavy chain alpha and beta isoforms as potential biomarkers.
Classifier peptides | ||
Identified protein | CHD disease | AVD disease |
Myosin heavy chain alpha isoform | KLAEKDEEMEQAK, | RKLEGDLK, |
NLQEEISDLTEQLGEGGKNVHELEKVR | AQLEFNQIK, | |
GSSFQTVSALHR, | ||
GKLSYTQQMEDLKR | ||
Myosin heavy chain beta isoform | KLAEKDEEMEQAK | RKLEGDLK, |
AQLEFNQIK, | ||
GSSFQTVSALHR |
Based on the thoughts above, a possible explanation for the different classifier peptides in CHD and AVDs leading to the same myosin may be differences in tryptic digestion patterns caused by posttranslational modifications at different positions. An investigation of the correlation to these differences is the topic of our ongoing work. For this particular study, fragmentations by electron capture dissociation (ECD) or electron transfer dissociation (ETD) are more suitable than collision-induced dissociation (CID), since ECD and ETD protect posttranslational modifications while breaking the peptide backbone bonds.
Another possible reason for altered digestion specificity of trypsin could be the folding geometry. If the protein is not completely unfolded during the digestion process, the tryptic cleavage at particular sites can be sterically hindered. This would then lead to missed cleavages.
A number of proteins in ESI-FT-ICR MS/MS were also identified with the method MALDI-TOF/TOF mass spectrometry with the same peptide sequences. In samples from the CHD group, commonly identified proteins were adenylate kinase, myosin heavy chain beta isoform. In samples from the AVD group, the proteins commonly identified in FT-ICR MS/MS and TOF/TOF were desmin, alpha cardiac actin, myosin heavy chain alpha isoform, myosin heavy chain beta isoform, and ADP/ATP translocase [
As described previously, FT-ICR and MALDI-TOF mass spectrometry used different methods of ionization. For the FT-ICR MS, ions were separated by liquid-chromatography and online ionized by electrospray ionization and transferred into mass spectrometer—either directly or after collision-induced dissociation—for detection. The MALDI-TOF mass spectrometric method uses samples in deposited solid phase. Thus, the components in the samples were LC-separated first and fractions were deposited on a MALDI sample plate precoated with a alpha-cyano-4-hydroxy cinnamic acid as a matrix (Pre Spotted Anchor Chip target. Bruker Daltonik, Bremen, Germany). This plate was inserted into the ion source of the TOF mass spectrometer, irradiated with the laser beam, and the ions were generated by matrix assisted laser desorption/ionization and detected. These ions are in general singly charged. Although compounds in both MALDI and electrospray ionization become mildly ionized, due to the different ionization techniques in MALDI-TOF and FT-ICR instruments, and to the non-online method in the LC-MALDI-TOF, some of the ions may have different abundances than in the on-line system. Thus, some of the FT-ICR MS/MS or MALDI-TOF/TOF dissociations of classifiers could not be performed as the abundance in the corresponding system was low. One of the examples is the alpha crystallin B chain [
As an overview, the bottom-up proteomic approach was applied to proteins in human cardiac muscle tissue samples from two groups of patients in this study. The selection of patients enabled the examination of two clearly separated case etiologies. Proteins in the tissue samples were digested by trypsin, and the digest containing a mixture of peptides was analyzed by mass spectrometry after liquid chromatographic separation. The use of high-resolution mass spectrometry (in this case FT-ICR MS) allowed to resolve and display the mass spectrometric peaks in this complex picture of the LC-MS results and to compare both separate disease forms. By comparison of LC-MS diagrams, the classifier masses could be clearly identified. These were selected in the subsequent experiments and fragmented (LC-FT-ICR MS/MS), in order to identify the proteins which had generated the classifier masses. The comparison clearly separated both disease forms while the analysis and identification of the proteins which led to the classifiers helped to study the biomarkers related to CHD and AVD. An additional work for comparing LC-MALDI-TOF mass spectrometry and LC-MALDI TOF/TOF for the MS/MS fragmentation has also be performed.
The unique patient selection in this study, combined with the bottom-up proteomic approach using liquid chromatography and high resolution mass spectrometry, seems to be highly efficient in determination of the differences between selected disease groups. The
This study of the heart muscle tissue samples helped establish a first picture of the proteomic appearance of two virtually independent etiologies of heart disease. As our main target is to diagnose cardiac disease less invasively and directly, we are currently investigating blood plasma samples from CHD and AVD patients in order to identify the differences with the same technique using differential high-resolution mass spectrometry.
The authors thank Sören Deininger and Arndt Asperger for their help with liquid-chromatography and sample deposition processes prior to MALDI-TOF and MALDI-TOF/TOF MS experiments. Financial support from the Swedish Research Council (Grant 621–2008-3562, 621-2011-4423, 342-2004-3944 (JB)) is gratefully acknowledged.