Systematic development of an enzymatic phosphorylation assay compatible with mass spectrometric detection

Abstract The enzymatic peptide phosphorylation by cAMP-dependent protein kinase A (PKA) was optimized and monitored by means of electrospray ionization mass spectrometry (ESI-MS). The direct detection of phosphorylated peptides by MS renders labeling unnecessary, reduces time and labor, due to less initial sample pretreatment. In this study the phosphorylation of the peptide malantide by PKA was performed in batch and reaction compounds were detected by ESI-MS after the incubation time. The subsequent product quantitation was accomplished by using one-point normalization. Applying this set-up, optimum solvent conditions (such as salt and modifier content), concentrations of essential reaction compounds (such as cAMP, Mg²⁺ and ATP), and the influence of reaction properties (such as pH and reaction time) were determined. The reaction milieu has to be suitable for both, the enzymatic reaction and the mass spectrometric detection. We found that the modifier content and the pH value had to be changed after the enzymatic reaction occurred. Through the addition of methanol and acetic acid, the reaction stopped immediately and a more sensitive mass spectrometric detection could be obtained simultaneously. Furthermore, an inhibitor study was performed, testing the inhibition potency of three protein kinase A inhibitors (PKIs). IC₅₀ values were determined and used to calculate the K _i values, that were 7.4, 19.0 and 340.0 nmol/L for PKI(6-22)amide, PKI(5-24)amide, and PKI(14-24)amide, respectively. These data vary between factor 4.4 (for PKI(6-22)amide) and 8.3 (for PKI(5-24)amide) compared to the K _i values described in literature. However, the K _i values are in good agreement with the data mainly obtained by fluorescence- or radioactivity-based methods. Nevertheless, our results indicate that ESI-MS is a realistic alternative to radioactivity and fluorescence detection in determining enzymatic activity. Furthermore we were able to illustrate its high potential as a quantitative detection method.     

Synthesis of thiol-modified peptide nucleic acids designed for post-assembly conjugation reactions

Two orthogonally protected PNA monomers were prepared having the mercaptomethyl moiety attached to the PNA backbone. These building blocks were employed in solid-phase PNA synthesis and it was shown that Boc/S-p-methoxybenzyl protection scheme was only satisfactory for the introduction of N-terminal thiol modification while the Fmoc/S-butylthio protected monomer proved to be amenable to elongation. The mercaptomethyl modification did not influence the thermal stability of a PNA/RNA duplex. The feasibility of PNA-PNA native ligation was demonstrated.     

功能多肽在肿瘤治疗中的应用及研究进展

多肽具有生物相容性好,功能多样化,生物体内响应性高及合成修饰方法简单易行等优点,已被广泛用于构建靶向药物传递系统。以具有靶向功能和刺激响应性的多肽为基础构建的药物传递系统,能够将药物定向地运送到肿瘤区域。药物传递系统到达肿瘤组织后,在肿瘤组织特殊微环境或外源刺激下,实现药物的精准释放。这种具有特异性肿瘤靶向和刺激响应型的多肽载体可以最大程度地提高药物的抗肿瘤效果,降低药物的毒副作用。本文简要介绍了常用的靶向多肽和刺激响应型多肽,并讨论了基于功能型多肽的药物载体在肿瘤治疗方面的应用。     

Cationic Graft Copolymers as Carriers for Delivery of Antisense‐Oligonucleotides

Self‐assembling systems based on ionic complexes of DNA fragments (36 base pairs), bcl‐2 antisense oligonucleotides (octadecamer), oligophosphates (25 phosphate groups) or acrylic oligomers (18 groups of phosphonic acid) with poly(L ‐lysine) (PLL) ( = 130 000 and 88 000) grafted with short poly[N‐(2‐hydroxypropyl)methacrylamide] (PHPMA) chains ( = 4 300 or 8 600) were studied by static and dynamic light scattering methods as systems suitable for gene therapy applications. The graft copolymers (GPLLs) with shorter PHPMA grafts ( = 4 300) provide polyelectrolyte complexes (PECs) with smaller and R_H than the corresponding GPLLs with longer grafts ( = 8 600) and the same content of PLL. The lowest aggregation number of 2 was observed for PECs prepared from the GPLL with short grafts and 40 wt.‐% of PLL. The complexes of oligonucleotides and DNA fragments with GPLLs showed quite similar behavior to that with oligophosphates and acrylic oligomer. The complexes prepared from GPLLs containing 40 wt.‐% of PLL and at excess of oligophosphate were stable for at least 48 h under physiological conditions (0.15 M NaCl) and in bovine serum albumin solutions (1 mg · mL^?1). Additionally, polyanion exchange reactions of the PECs in contact with poly(styrenesulfonate) and DNA were studied in 0.15 M NaCl solutions. The oligophosphates in complexes were at least partially substituted with high‐molecular‐weight polyanions. The structure of the initial PECs dominated the PEC structure after the exchange reaction.

The dependence of the molecular weight (a) and the hydrodynamic radius R_H (b) of complexes of the oligophosphate (OPP) and four graft copolymers (GPLLi, i = 0–3) on the mixing ratio X.     

Peptide dissociation in solution or bound to a polymer: comparative solvent effect

Dissociation of peptide when in solution or attached to a polymer was investigated. Magnified solvation of peptide-resins occurred in solvent with similar polarity. Conversely the solubilization of peptides was not usually directly related to the medium polarity. The greater the difference between acidity and basicity of solvent and its potential to form van der Waals interaction, the stronger its solubilization strength. Solvents with similar electrophilicity and nucleophilicity usually did not solvate aggregated peptide-resins nor dissolve peptides. The peptide solubilization in water-containing mixed solvents depended on combination of acidity/basicity of both components. Some criteria for choosing suitable solvents for peptide-resin solvation or peptide solubilization could be advanced.     

A supramolecular co-delivery strategy for combined breast cancer treatment and metastasis prevention

We herein propose a co-delivery approach where small interference RNA (siRNA) and anticancer chemotherapeutic drug are simultaneously loaded into a single delivery carrier for the combined treatment of breast cancer and metastasis prevention. The co-delivery vector is composed of chondroitin sulfate (CS)-coated β-cyclodextrin-polyethylenemine polymer, which is capable of loading paclitaxel (PTX) and siRNA simultaneously to form therapeutic nanocomplexes. The nanocomplex, termed as CP-PTX-siCD146-CS, is demonstrated to have strong active targeting ability towards CD44-overexpresing breast cancer cells. Moreover, the co-delivery of PTX and siRNA not only effectively inhibits cancer cells proliferation and induces apoptosis, but also well prevents metastasis. Importantly, CP-PTX-siCD146-CS nanocomplexes exhibit stronger cytotoxic effects and anti-metastatic effects on MBA-MD-231 breast cancer cells, in comparison with PTX or siCD146 mono-treatment. The current study defines a potential therapeutic strategy for the combined breast cancer treatment and metastasis prevention from a co-delivery perspective.     

Discovery of carboxypeptidase Y as a catalyst for the incorporation of sterically demanding α-fluoroalkyl amino acids into peptides

The first example of a direct enzymatic coupling of two different, sterically demanding C^α-fluoroalkyl amino acids to amino acid nucleophiles is reported. N-Protected Ala methyl ester derivatives bearing a methyl-, difluoromethyl-, or trifluoromethyl group, respectively, instead of the α-proton were accepted as substrates by carboxypeptidase Y and could, therefore, be coupled directly to various nucleophiles.     

Multiplex multidimensional nanoLC-MS system for targeted proteomic analyses

The present work describes a dual-column and dual-sprayer LC-MS system for high-throughput proteomic analyses. This system consists of two precolumns for sample desalting and two analytical columns. Each column is terminated by a nanoelectrospray emitter mounted on a robotic arm enabling their sequential positioning in front of the sampling cone of the mass spectrometer. The effluent from each emitter is recorded in separate acquisition channels without detectable crosstalk. Gradient elution to both nanoLC columns is delivered by a single HPLC system via a flow splitter. The reproducibility of retention time and peak intensity of the present multiplex system were comparable to those obtainable using a single emitter configuration. Replicate injections of complex tryptic digests (n = 10) indicated that this system provided good reproducibility of retention time and peak intensity on both columns with RSD values of less than 0.9 and 18.6%, respectively. The application of this system is demonstrated for the monitoring of protein expression changes in U937 human monocyte cells with and without phorbol ester administration. Furthermore, we also demonstrated the use of this multiplex system in a 2-D LC configuration to increase sample loading and throughput for the analysis of biomarker samples of higher complexity. Variations in peptide abundance down to two-fold change were identified across salt fractions for spiked tryptic digests present at a level of 50 fmol in 1.5 microg of plasma samples.     

蛋白质全新设计：八残基序列形成发夹结构的圆二色谱

β-发夹是天然蛋白质中丰富的二级结构单元之一，在蛋白折叠和功能方面扮演着重要角色.文章报导了二条多肽序列（LTVd-PGLTV，n7和 LTVGDDTV， n5）的设计、合成和园二色谱研究结果.结果显示，n5在198 nm附近呈现负峰，表现为非规整结构特征；相反，n7表现为典型的发夹结构特征，在218 nm附近呈负峰,196 nm附近呈正峰，为β-转角与β-折叠的共同贡献.初步研究表明，β-转角、序列关系和氨基酸形成β-折叠结构倾向性是β-发夹结构形成和稳定的决定性因素.     

Application of Electrochemiluminescence Sensor to a Rapid Method of Estimating Activity of Enzyme in Hydrolysis of Peptides

An advanced flow‐through electrochemiluminescence (ECL) detector was applied to determination of resulting amino acids and oligopeptides in hydrolysis of four peptides by leucine aminopeptidase. For 20mer polypeptide PRGPDRPEGIEEEGGERDRD, the ECL intensity due to the polypeptide and produced proline was analyzed to give the dissociation contant K_m (1.2×10^?4 M) and the catalytic reaction constant k_cat (3.7 s^?1), which were good agreement with those obtained by HPLC. The enzymatic parameters were similarly obtained for the other peptides.