RESUMEN
Protein phosphorylation is a crucial post-translational modification that plays an important role in the regulation of cellular signaling processes. Site-specific quantitation of phosphorylation levels can help decipher the physiological functions of phosphorylation modifications under diverse physiological statuses. However, quantitative analysis of protein phosphorylation degrees is still a challenging task due to its dynamic nature and the lack of an internal standard simultaneously available for the samples differently prepared for various phosphorylation extents. In this study, stable-isotope dimethyl labeling coupled with phosphatase dephosphorylation (DM + deP) was tried to determine the site-specific degrees of phosphorylation in proteins. Firstly, quantitation accuracy of the (DM + deP) approach was confirmed using synthetic peptides of various simulated phosphorylation degrees. Afterwards, it was applied to evaluate the phosphorylation stoichiometry of milk caseins. The phosphorylation degree of Ser130 on α-S1-casein was also validated by absolute quantification with the corresponding synthetic phosphorylated and nonphosphorylated peptides under a selected reaction monitoring (SRM) mode. Moreover, this (DM + deP) method was used to detect the phosphorylation degree change of Ser82 on the Hsp27 protein of HepG2 cells caused by tert-butyl hydroperoxide (t-BHP) treatment. The results showed that the absolute phosphorylation degree obtained from the (DM + deP) approach was comparable with the relative quantitation resulting from stable-isotope dimethyl labeling coupled with TiO2 enrichment. This study suggested that the (DM + deP) approach is promising for absolute quantification of site-specific degrees of phosphorylation in proteins, and it may provide more convincing information than the relative quantification method.
Asunto(s)
Marcaje Isotópico , Monoéster Fosfórico Hidrolasas/metabolismo , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Caseínas/química , Cromatografía Liquida , Proteínas de Choque Térmico HSP27/metabolismo , Células Hep G2 , Humanos , Péptidos/química , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Reproducibilidad de los Resultados , terc-Butilhidroperóxido/farmacologíaRESUMEN
In this study, we screened 10 resveratrol derivatives isolated from Ampelopsis brevipedunculata var. hancei (Planch.) Rehder (ABH) for angiotensin I converting enzyme (ACE) inhibitory (ACEI) activity. Among these compounds, (+)-hopeaphenol and (+)-vitisin A showed the lowest IC50 values (â¼ 1.5 µM) toward ACE. In addition, the compounds' abundances and distributions in ABH were profiled using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Interestingly, trimers and tetramers of resveratrol were mainly obtained from the bark of ABH when 90% ethanol was used for extraction. This result implies that the antihypertension effect of ABH extract may be mainly contributed by (+)-hopeaphenol (F1) and (+)-vitisin A (F2) in the ABH bark due to their remarkable ACE inhibitions. Moreover, the sizes and structures of these compounds were further correlated to their affinities toward ACE using molecular docking calculations. The results showed that resveratrol tetramers interact with ACE more favorably than other smaller oligomers.
Asunto(s)
Ampelopsis/química , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Productos Biológicos/farmacología , Estilbenos/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/química , Inhibidores de la Enzima Convertidora de Angiotensina/aislamiento & purificación , Productos Biológicos/administración & dosificación , Productos Biológicos/aislamiento & purificación , Cromatografía Liquida/métodos , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Resveratrol , Estilbenos/administración & dosificación , Estilbenos/aislamiento & purificación , Espectrometría de Masas en Tándem/métodosRESUMEN
In this study, a novel angiotensin-converting enzyme (ACE)-inhibitory tripeptide (IVR) was isolated and identified from unfertilized soft-shelled turtle egg white (SSTEW). The IC50 value of IVR was measured in vitro as low as 0.81 ± 0.03 µM, and its inhibition type was suggested as competitive according to the Lineweaver-Burk plot. This peptide can be generated from either thermolysin followed by trypsin digestion (two stages) or only trypsin digestion (one stage). Quantitative LC-MS/MS analysis indicated that two-stage digestion gave 3.14 ± 0.17 mg of IVR from 1 g of SSTEW, better than that from one-stage digestion (1.31 ± 0.12 mg). In vivo antihypertensive activity of the tripeptide IVR after single oral administration (0.1 and 1 mg/kg of body weight) led to a significant reduction in systolic blood pressure 2-4 h after administration in spontaneously hypertensive rats. In addition, the binding mechanism of IVR has been rationalized through docking simulations using the testicular ACE (tACE)-lisinopril complex at 2 Å resolution (PDB 108A ). The best docking pose was located at the tACE catalytic site resembling the mode of inhibition exerted by lisinopril, an effective hypertensive synthetic drug. The degree of inhibition of this peptide correlated with the H-bond interaction between the C-terminal of IVR and Lys511 and Tyr520 residues of tACE, a significant inhibitor registration for lisinopril. This study illustrated that IVR behaves as a transition-state analogue inhibitor and is useful in therapeutic intervention for blood pressure control. To the best of our knowledge, this is the first report of an efficient ACE-inhibitory tripeptide generated from the unfertilized egg of soft-shelled turtle.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/química , Inhibidores de la Enzima Convertidora de Angiotensina/aislamiento & purificación , Antihipertensivos/química , Antihipertensivos/aislamiento & purificación , Clara de Huevo/química , Péptidos/química , Péptidos/aislamiento & purificación , Inhibidores de la Enzima Convertidora de Angiotensina/administración & dosificación , Animales , Antihipertensivos/administración & dosificación , Biocatálisis , Presión Sanguínea , Humanos , Hipertensión/tratamiento farmacológico , Hipertensión/fisiopatología , Cinética , Masculino , Simulación del Acoplamiento Molecular , Péptidos/administración & dosificación , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/metabolismo , Ratas , Ratas Endogámicas SHR , Tripsina/química , TortugasRESUMEN
Protein N-terminal acetylation is one of the most common modifications occurring co- and post-translationally on either eukaryote or prokaryote proteins. However, compared to other protein modifications, the physiological role of protein N-terminal acetylation is relatively unclear. To explore the biological functions of protein N-terminal acetylation, a robust and large-scale method for qualitative and quantitative analysis of this modification is required. Enrichment of N(α)-acetylated peptides or depletion of the free N-terminal and internal tryptic peptides prior to analysis by mass spectrometry are necessary based on current technologies. This study demonstrated a simple strong cation exchange (SCX) fractionation method to selectively enrich N(α)-acetylated tryptic peptides via dimethyl labeling without the need for tedious protective labeling and depleting procedures. This method was introduced for the comprehensive analysis of N-terminal acetylated proteins from HepG2 cells. Several hundred N-terminal acetylation sites were readily identified in a single SCX flow-through fraction. Moreover, the N(α)-acetylated peptides of some protein isoforms were simultaneously observed in the SCX flow-through fraction, which indicated that this approach can be utilized to discriminate protein isoforms with very similar full sequences but different N-terminal sequences, such as ß-actin/γ-actin, ERK1/ERK2, α-centractin/ß-centractin, and ADP/ATP translocase 2 and 3. Compared to other methods, this method is relatively simple and can be directly implemented in a two-dimensional separation (SCX-RP)-mass spectrometry scheme for quantitative N-terminal proteomics using stable-isotope dimethyl labeling.