RESUMO
The complexation behavior of a series of paraquats (G1.2PF(6)-G5.2PF(6)) and bis(pyridinium) derivatives (G6.2PF(6)-G14.2PF(6)) with pillar[5]arene (P5A) host has been comprehensively investigated by (1)H NMR, ESI mass and UV-vis absorption spectroscopy. It is found that P5A forms 2 : 1 external complexes with N,N'-dialkyl-4,4'-bipyridiniums (G1-G4.2PF(6)); while it forms 1 : 1 pseudorotaxane-type inclusion complexes with methylene [-(CH(2))(n)-] linked bis(pyridinium) derivatives possessing appropriate chain lengths (n = 3-6, G7-G10.2PF(6)). Host-guest association constants in dimethyl sulfoxide (DMSO) were determined, indicating G7-G10.2PF(6) axles form stable [2]pseudorotaxanes with P5A wheel in this very high polarity solvent and 1,4-bis(pyridinium)butane (G8.2PF(6)) was the most suitable axle unit. Meanwhile, the nature of the substituents attached to 1,4-bis(pyridinium)butane dramatically affects the molecular recognition behavior. The introduction of pyridyls (G13.2PF(6)) increases not only the K(a) value (4.5 x 10(2) --> 7.4 x 10(2) M(-1)), but also the charge transfer (CT) absorption (colorless --> yellow). Furthermore, the solvent effects have also been investigated, showing they significantly influence the association strength during the course of host-guest complexation. Particularly, the K(a) value of P5A-G13.2PF(6) in 1 : 1 (v:v) acetone-d(6)/DMSO-d(6) is enhanced by a factor of 7.3 compared with pure DMSO-d6 (7.4 x 10(2) --> 5.4 x 10(3) M(-1)).
RESUMO
BACKGROUND: Lysine acetylation is a reversible and dynamic post-translational modification on proteins, and plays an important role in diverse biological processes. Technological limitations have so far prevented comparative quantification of lysine acetylation in different samples. RESULTS: We developed a method to efficiently study lysine acetylation on individual proteins from complex mixtures, using antibody microarrays to capture individual proteins followed by detection with lysine acetyl antibody. By profiling both protein and acetylation variations in multiple samples using this microarray, we found cancer-associated lysine acetylation alteration on VEGF in the serum of hepatocellular carcinoma patients. CONCLUSION: Microarrays of lysine acetylation are highly effective for detecting acetylation, and should be useful in identifying and validating disease-associated acetylation alterations as biomarkers under both normal and pathological circumstances.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/sangue , Neoplasias Hepáticas/sangue , Análise Serial de Proteínas/métodos , Processamento de Proteína Pós-Traducional , Fator A de Crescimento do Endotélio Vascular/genética , Acetilação , Animais , Anticorpos/química , Biomarcadores Tumorais/sangue , Biotina/química , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , Bovinos , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Lisina/metabolismo , Análise Serial de Proteínas/instrumentação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Soroalbumina Bovina/química , Estreptavidina/química , Fator A de Crescimento do Endotélio Vascular/sangueRESUMO
Trypsin was covalently immobilized to graphene oxide (GO)-poly(urea-formaldehyde) (PUF) composite coated on the channel wall of poly(methyl methacrylate) microchips to fabricate microfluidic bioreactors for highly efficient proteolysis. A mixture solution containing urea-formaldehyde prepolymer and GO nanosheets was allowed to flow through the channels. The modification layer on the channel wall could further polycondense to form GO-PUF composite coating in the presence of ammonium chloride. The primary amino groups of trypsin could react with the carboxyl groups of the GO sheets in the coating with the aid of carboxyl activating agents to realize covalent immobilization. The feasibility and performance of the novel GO-based microchip bioreactors were demonstrated by the digestion of bovine serum albumin, lysozyme, ovalbumin, and myoglobin. The digestion time was significantly reduced to less than 5s. The obtained digests were identified by MALDI-TOF MS with satisfactory sequence coverages that were comparable to those obtained by using 12-h in-solution digestion. The present proteolysis strategy is simple and efficient, offering great promise for high-throughput protein identification.