RESUMEN
Atherogenic cardiovascular diseases are the major cause of mortality. Prevention and prediction of incidents is important; however, biomarkers that directly reflect the disease progression remain poorly investigated. To elucidate molecular determinants of atherogenesis, proteomic approaches are advantageous by using model animals for comparing changes occurring systematically (bloodstream) and locally (lesion) in accordance with the disease progression stages. We conducted differential mass spectrometric analysis between apolipoprotein E deficient (apoED) and wild-type (wt) mice using the plasma and arterial tissue of both types of mice obtained at four pathognomonic time points of the disease. A total of 100 proteins in the plasma and 390 in the arterial tissues were continuously detected throughout the four time points; 29 were identified in common. Of those, 13 proteins in the plasma and 36 in the arterial tissues showed significant difference in abundance between the apoED and wt mice at certain time points. Importantly, we found that quantitative variation patterns regarding the pathognomonic time points did not always correspond between the plasma and arterial tissues, resulting in gaining insight into atherosclerotic plaque progression. These characteristic proteins were found to be components of inflammation, thrombus formation, and vascular remodeling, suggesting drastic and integrative alteration in accordance with atherosclerosis development.
Asunto(s)
Arterias/química , Aterosclerosis/metabolismo , Proteínas Sanguíneas/aislamiento & purificación , Placa Aterosclerótica/metabolismo , Trombosis/metabolismo , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Arterias/metabolismo , Arterias/patología , Aterosclerosis/genética , Aterosclerosis/patología , Biomarcadores/sangre , Cromatografía Liquida , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación de la Expresión Génica , Inflamación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Anotación de Secuencia Molecular , Fragmentos de Péptidos/análisis , Placa Aterosclerótica/genética , Placa Aterosclerótica/patología , Proteolisis , Coloración y Etiquetado , Espectrometría de Masas en Tándem , Trombosis/genética , Trombosis/patología , Tripsina , Remodelación VascularRESUMEN
We developed a liquid chromatography (LC) compatible electron capture dissociation (ECD) mass spectrometer for glycoproteomics, with which ECD and hot ECD (HECD) experiments can be flexibly switched by quickly changing the electron energy without further tuning of the mass spectrometer. Desialylated glycopeptides were dissociated well in both ECD and HECD experiments. For sialylated glycopeptides, on the other hand, ECD with electron energy higher than 4 eV showed significantly higher sequence coverage than that with an electron energy of 0.2 eV. A nano LC system was coupled to our ECD mass spectrometer to investigate N-linked glycopeptides from lysylendopeptidase (Lys-C) digests of human transferrin. ECD spectra at multiple electron energies of 0.2, 5.0, and 9.0 eV were obtained for each targeting precursor ion in a single LC injection. Glycopeptides with a sialylated bi-, tri-, or tetra-antennary complex N-glycan were identified with high sequence coverage by HECD. Glycopeptides with tri- or tetra-antennary N-glycans have seldom been analyzed by ECD or ETD before this report. We also found that a preferential dissociation of nonreducing termini of glycans in glycopeptides by ECD and HECD.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Glicopéptidos/análisis , Espectrometría de Masas en Tándem , Secuencia de Aminoácidos , Animales , Pollos , Cromatografía Líquida de Alta Presión/instrumentación , Proteínas del Huevo/metabolismo , Glicopéptidos/química , Humanos , Iones/química , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Transferrinas/metabolismoRESUMEN
RATIONALE: Electron capture dissociation (ECD) is useful tool for sequencing of peptides and proteins with post-translational modifications. To increase the sequence coverage for peptides and proteins, it is important to develop ECD device with high fragmentation efficiency. METHODS: Sequence analysis of intact undigested bioactive peptides (3000-5000 Da) was performed by use of electron capture dissociation (rf-ECD) and collision-induced dissociation (CID) in a linear radio-frequency quadrupole ion trap that was coupled to a time-of-flight mass spectrometer. We applied rf-ECD, hot rf-ECD (rf-ECD with high electron energy), and CID for intact bioactive peptide ions of various charge states and evaluated the sequence coverage of their fragment spectra. RESULTS: Hot rf-ECD produced a higher number of c- and z-type fragment ions of modified peptide ions as electron energy increased in lower charged peptide ions, and sequence coverage greater than 80% was obtained compared with the CID case (40-80%). CONCLUSIONS: The result indicates that intact bioactive modified peptides (Ghrelin, ANP) were correctly identified by use of hot rf-ECD.
Asunto(s)
Espectrometría de Masas/métodos , Péptidos/química , Proteínas/química , Secuencia de Aminoácidos , Animales , Humanos , Espectrometría de Masas/instrumentación , Datos de Secuencia Molecular , Mapeo Peptídico , Péptidos/genética , Proteínas/genética , RatasRESUMEN
Despite the growing importance of mucin core O-glycosylation in many biological processes including the protection of epithelial cell surfaces, the immune response, cell adhesion, inflammation, and tumorigenesis/metastasis, the regulation mechanism and conformational significance of the multiple introduction of α-GalNAc residues by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs) remains unclear. Here we report an efficient approach by combining MS and NMR spectroscopy that allows for the identification of O-glycosylation site(s) and the effect of O-glycosylation on the peptide backbone structures during enzymatic mucin domain assembly by using an isoform UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-T2 (ppGalNAcT2) in vitro. An electron-capture dissociation device in a linear radio-frequency quadrupole ion trap (RFQ-ECD) combined with a time-of-flight (TOF) mass spectrometer was employed for the identification of Thr/Ser residues occupied by α-GalNAc branching among multiple and potential O-glycosylation sites in the tandem repeats of human mucin glycoproteins MUC4 (Thr-Ser-Ser-Ala-Ser-Thr-Gly-His-Ala-Thr-Pro-Leu-Pro-Val-Thr-Asp) and MUC5AC (Pro-Thr-Thr-Val-Gly-Ser-Thr-Thr-Val-Gly). In the present study, O-glycosylation was initiated specifically at Thr10 in naked MUC4 peptide and additional introduction of α-GalNAc proceeded preferentially but randomly at three other Thr residues to afford densely glycosylated MUC4 containing six α-GalNAc residues at Thr1, Ser2, Ser5, Thr6, Thr10, and Thr15. On the contrary, O-glycosylation of naked MUC5AC peptide occurred predominantly at consecutive Thr residues and led to MUC5AC with four α-GalNAc residues at Thr2, Thr3, Thr7, and Thr8. The solution structures determined by NMR spectroscopic studies elicited that the preferential introduction of α-GalNAc at Thr10 of MUC4 stabilizes specifically a ß-like extended backbone structure at this area, whereas other synthetic models with a single α-GalNAc residue at Thr1, Thr6, or Thr15 did not exhibit any converged three-dimensional structure at the proximal peptide moiety. Such conformational impact on the underlying peptides was proved to be remarkable in the glycosylation at the consecutive Thr residues of MUC5AC.
Asunto(s)
Glicopéptidos/química , Mucina 5AC/química , Mucina 4/química , Mucinas/química , N-Acetilgalactosaminiltransferasas/metabolismo , Secuencia de Aminoácidos , Glicopéptidos/metabolismo , Glicosilación , Humanos , Modelos Moleculares , Mucinas/síntesis química , Mucinas/metabolismo , Resonancia Magnética Nuclear Biomolecular , Serina/química , Treonina/químicaRESUMEN
A mass-spectrometric method for a de novo determination of O-glycosylation heterogeneity was developed. We used a mild fragmentation technique, electron capture dissociation (ECD), which enables the determination of glycosylation sites as well as peptide sequencing. To demonstrate the correct identification of glycopeptides, we prepared a series of glycopeptides with the same peptide sequence and 6 different glycan modifications. ECD spectra were obtained at various electron energies, and were analyzed with the Mascot database-search engine. The obtained candidate glycopeptides were further validated by confirming the spectral overlap of ECD fragment peaks with the theoretical peaks. The results indicate that all glycopeptides were unambiguously identified, including glycosylation sites by combining ECD results with different electron energies for each glycopeptide.