RESUMEN
The field of cell-penetrating peptides is dominated by the use of oligomers of arginine residues. Octanol-water partitioning in the presence of an anionic lipid is a validated proxy for cell-penetrative efficacy. Here, we add one, two, or three N-methyl groups to Ac-Arg-NH2 and examine the effects on octanol-water partitioning. In the absence of an anionic lipid, none of these arginine derivatives can be detected in the octanol layer. In the presence of sodium dodecanoate, however, increasing N-methylation correlates with increasing partitioning into octanol, which is predictive of higher cell-penetrative ability. We then evaluated fully Nα -methylated oligoarginine peptides and observed an increase in their cellular penetration compared with canonical oligoarginine peptides in some contexts. These findings indicate that a simple modification, Nα -methylation, can enhance the performance of cell-penetrating peptides.
Asunto(s)
Péptidos de Penetración Celular , Péptidos de Penetración Celular/química , Arginina/química , Metilación , Octanoles/química , Agua/química , LípidosRESUMEN
A method was developed for determination of proteinaceous free amino acids by gas chromatography-mass spectrometry (GC-MS). The guanidino group of arginine in amino acid samples was modified with 1,2-cyclohexanedione at room temperature under basic conditions, and all amino acids were directly derivatized with isobutyl chloroformate. The amino acid derivatives formed were analyzed by GC-MS. The method developed was applied successfully for the determination of amino acids in the Japanese alcoholic beverage, sake.
Asunto(s)
Aminoácidos/análisis , Vino/análisis , Etanol/química , Formiatos/química , Cromatografía de Gases y Espectrometría de Masas , JapónRESUMEN
The present work focused on the high-throughput screening and quantitation of guanidino compounds (GCs) and ureido compounds (UCs) in human thyroid tissues. The strategy employed benzylic rearrangement stable isotope labeling (BRSIL) for the sample preparation and then detection using liquid chromatography-drift tube ion mobility spectrometry-quadrupole time of flight mass spectrometry (LC-DTIMS-QTOF MS). A short reversed-phase LC realized an on-line desalting and a measurement cycle of 5.0 min. DTIMS separation enhanced the better specificity and selectivity for the benzil labeled GCs and UCs. The elevated mass resolution of QTOF MS enabled measure of the characteristic ions at accurate mass in MS and tandem MS spectra. Collision cross section (CCS) from DTIMS and accurate mass from QTOF MS were used as two qualifiers for the profiling and identification of GCs and UCs. In addition, an integral abundance arising from 3-D ion features (retention time, drift time, m/z) was applied to quantify the GCs and UCs in human thyroid tissues. The quantitative validation indicated good linearity (coefficient values ≥ 0.9981), good precision (1.0%-12.3% for intra-day and 0.9%-7.8% for inter-day) and good accuracy (91%-109%). The results demonstrated that the developed BRSIL coupled with LC-DTIMS-QTOF MS can be a powerful analysis platform to investigate GCs and UCs in human thyroid tissues.
Asunto(s)
Guanidinas/análisis , Ensayos Analíticos de Alto Rendimiento , Glándula Tiroides/química , Urea/análogos & derivados , Cromatografía Liquida , Humanos , Espectrometría de Movilidad Iónica , Marcaje IsotópicoRESUMEN
Benzylic rearrangement stable isotope labeling (BRSIL) was explored to quantify the guanidino and ureido compounds (GCs and UCs). This method employed a common reagent, benzil, to label the guanidino and ureido groups through nucleophilic attacking then benzylic migrating. The use of BRSIL was investigated in the analysis of five GCs (creatine, l-arginine, homoarginine, 4-guanidinobutyric acid, and methylguanidine) and two UCs (urea and citrulline). The labeling was found simple and specific. The introduction of bi-phenyl group and the generation of nitrogen heterocyclic ring in the benzil-d0/d5 labeled GCs and UCs improved the retention behaviors in liquid chromatography (LC) and increased the sensitivity of electrospray ionization mass spectrometry (ESI MS) detection. The fragment ion pairs of m/z 182/187 and m/z 210/215 from the benzil-d0/d5 tags facilitated the discovery of potential GCs and UCs candidates residing in biological matrices. The use of BRSIL combined with LC-ESI MS was applied for simultaneously quantitation of GCs and UCs in thyroid tissues. It was demonstrated that nine GCs and UCs were detected, six of which were further quantified based on corresponding standards. It was concluded that five GCs and UCs (l-arginine, homoarginine, 4-guanidinobutyric acid, methylguanidine, and citrulline) were statistically significantly different (p < 0.05) between the para-carcinoma and carcinoma thyroid tissue samples.
Asunto(s)
Cromatografía Liquida/métodos , Guanidinas/análisis , Marcaje Isotópico , Espectrometría de Masa por Ionización de Electrospray/métodos , Glándula Tiroides/química , Urea/análisisRESUMEN
A rigid bis(choloyl) conjugate functionalized with guanidino groups was synthesized and fully characterized on the basis of NMR ((1)H and (13)C) and ESI MS (LR and HR) data. Its transmembrane ionophoric activity across egg-yolk l-α-phosphatidylcholine-based liposomal membranes was investigated by means of chloride ion selective electrode technique and pH discharge assay. The data indicate that under the assay conditions, this conjugate was capable of promoting the transport of anions, presumably via a cation/anion symport process. A Hill analysis reveals that two molecules of this compound are assembled into the transport-active species.