RESUMEN
Linker stability is critically important for the efficacy and safety of peptide and protein conjugates used for biological applications. One common conjugation strategy, thiol-maleimide coupling, generates a succinimidyl thioether linker with limited stability under physiological conditions. We have shown in previous work that when a peptide with an N-terminal cysteine is conjugated to a maleimide reagent, a thiazine structure is formed via a chemical rearrangement. Our preliminary work indicated that the thiazine linker has favorable stability. Here, we report the evaluation of a thiazine linker as an alternative to the widely used succinimidyl thioether linker for thiol-maleimide bioconjugation. The stability of the thiazine conjugate in comparison to the thioether conjugate was assessed across a broad pH range. Additionally, the propensity for retro-Michael reaction and cross-reactivity with other thiols was evaluated by treating conjugates in the presence of glutathione. The studies indicated that the thiazine linker degrades markedly slower than the thioether conjugate. In addition, the thiazine linker is over 20 times less susceptible to glutathione adduct formation. The NMR study of the thiazine structure confirmed that the formation of the thiazine linker is a stereoselective process that yields a single diastereomer. In summary, we propose the use of the thiazine linker obtained by conjugation of maleimide-containing reagents with peptides or proteins presenting an N-terminal cysteine as a novel approach for bioconjugation. The advantages of this approach are the formation of a linker with a well-defined stereochemical configuration, increased stability at physiological pH, and a strongly reduced propensity for thiol exchange.
Asunto(s)
Cisteína , Compuestos de Sulfhidrilo , Compuestos de Sulfhidrilo/química , Maleimidas/química , Péptidos , GlutatiónRESUMEN
Thiazine formation during the conjugation of N-terminal cysteine peptides to maleimides is an underreported side reaction in the peptide literature. When the conjugation was performed at neutral and basic pH, we observed the thiazine isomer as a significant by-product. Nuclear magnetic resonance (NMR) spectroscopy confirmed the structure of the six-membered thiazine and ultra-high performance liquid chromatography (UHPLC) combined with tandem mass spectrometry (MS/MS) allowed for facile, unambiguous detection due to a unique thiazine mass fragment. Furthermore, substitution of various amino acids adjacent to the N-terminal cysteine in a tripeptide model system resulted in different rates of thiazine formation, albeit within the same order of magnitude. We also determined that varying the N-substitution of the maleimide affects the thiazine conversion rate. Altogether, our findings suggest that thiazine rearrangement for N-terminal cysteine-maleimide adducts is a general side reaction that is applicable to other peptide or protein systems. Performing the conjugation reaction under acidic conditions or avoiding the use of an N-terminal cysteine with a free amino group prevents the formation of the thiazine impurity.
Asunto(s)
Cisteína/química , Maleimidas/química , Péptidos/química , Tiazinas/química , Concentración de Iones de Hidrógeno , Estructura MolecularRESUMEN
[reaction: see text] A novel strategy to generate thioester peptides compatible with Fmoc chemistry is presented. Peptide-C(alpha)oxy-(2-mercapto-1-carboxyamide)ethyl ester undergoes an O to S acyl shift during ligation and the newly formed thioester intermediate reacts with an N-terminal cysteine fragment generating a product with native amide bond at the ligation site.
Asunto(s)
Oxígeno/química , Péptidos/síntesis química , Azufre/química , Ésteres del Ácido Sulfúrico/síntesis química , Amidas/química , Cromatografía Líquida de Alta Presión , Cisteína/química , Concentración de Iones de Hidrógeno , Ligandos , Estructura Molecular , Péptidos/química , Sensibilidad y Especificidad , Ésteres del Ácido Sulfúrico/química , Factores de TiempoRESUMEN
We present an integrated proteomics platform designed for performing differential analyses. Since reproducible results are essential for comparative studies, we explain how we improved reproducibility at every step of our laboratory processes, e.g. by taking advantage of the powerful laboratory information management system we developed. The differential capacity of our platform is validated by detecting known markers in a real sample and by a spiking experiment. We introduce an innovative two-dimensional (2-D) plot for displaying identification results combined with chromatographic data. This 2-D plot is very convenient for detecting differential proteins. We also adapt standard multivariate statistical techniques to show that peptide identification scores can be used for reliable and sensitive differential studies. The interest of the protein separation approach we generally apply is justified by numerous statistics, complemented by a comparison with a simple shotgun analysis performed on a small volume sample. By introducing an automatic integration step after mass spectrometry data identification, we are able to search numerous databases systematically, including the human genome and expressed sequence tags. Finally, we explain how rigorous data processing can be combined with the work of human experts to set high quality standards, and hence obtain reliable (false positive < 0.35%) and nonredundant protein identifications.
Asunto(s)
Líquidos Corporales/química , Perfilación de la Expresión Génica , Gestión de la Información/métodos , Proteínas/análisis , Proteínas/química , Proteómica/métodos , Cromatografía/instrumentación , Cromatografía/métodos , Biología Computacional , Bases de Datos Factuales , Humanos , Gestión de la Información/instrumentación , Espectrometría de Masas/instrumentación , Espectrometría de Masas/métodos , Péptidos/análisis , Proteínas/genética , Proteínas/metabolismo , Reproducibilidad de los Resultados , Interfaz Usuario-ComputadorRESUMEN
Human blood plasma is a useful source of proteins associated with both health and disease. Analysis of human blood plasma is a challenge due to the large number of peptides and proteins present and the very wide range of concentrations. In order to identify as many proteins as possible for subsequent comparative studies, we developed an industrial-scale (2.5 liter) approach involving sample pooling for the analysis of smaller proteins (M(r) generally < ca. 40 000 and some fragments of very large proteins). Plasma from healthy males was depleted of abundant proteins (albumin and IgG), then smaller proteins and polypeptides were separated into 12 960 fractions by chromatographic techniques. Analysis of proteins and polypeptides was performed by mass spectrometry prior to and after enzymatic digestion. Thousands of peptide identifications were made, permitting the identification of 502 different proteins and polypeptides from a single pool, 405 of which are listed here. The numbers refer to chromatographically separable polypeptide entities present prior to digestion. Combining results from studies with other plasma pools we have identified over 700 different proteins and polypeptides in plasma. Relatively low abundance proteins such as leptin and ghrelin and peptides such as bradykinin, all invisible to two-dimensional gel technology, were clearly identified. Proteins of interest were synthesized by chemical methods for bioassays. We believe that this is the first time that the small proteins in human blood plasma have been separated and analyzed so extensively.
Asunto(s)
Análisis Químico de la Sangre/métodos , Proteínas Sanguíneas/metabolismo , Plasma/metabolismo , Proteómica/métodos , Secuencia de Aminoácidos , Cromatografía , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Biología Computacional , Bases de Datos como Asunto , Electroforesis en Gel Bidimensional/métodos , Humanos , Inmunoglobulina G/química , Espectrometría de Masas , Datos de Secuencia Molecular , Péptidos/química , Proteoma , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Fracciones Subcelulares , Factores de Tiempo , Tripsina/farmacologíaRESUMEN
Airway inflammation is a characteristic feature in airway diseases such as asthma and chronic obstructive pulmonary disease. Oxidative stress, caused by the excessive production of reactive oxygen species by inflammatory cells like macrophages, eosinophils and neutrophils, is thought to be important in the complex pathogenesis of such airway diseases. The calcium-sensing regulatory protein calmodulin (CaM) binds and regulates different target enzymes and proteins, including calcium channels. In the present study, we investigated whether CaM, via the modulation of calcium channel function, influences [Ca(2+)](i) in pulmonary inflammatory cells, and consequently, modulates the production of reactive oxygen species by these cells. This was tested with a peptide termed calcium-like peptide 2 (CALP2), which was previously shown to regulate such channels. Specifically, radical production by purified broncho-alveolar lavage cells from guinea-pigs in response to CALP2 was measured. CALP2 was a strong activator of alveolar macrophages. In contrast, CALP2 was only a mild activator of neutrophils and did not induce radical production by eosinophils. The CALP2-induced radical production was mainly intracellular, and was completely blocked by the NADPH-oxidase inhibitor DPI, the superoxide inhibitor SOD and the CaM antagonist W7. Furthermore, the calcium channel blocker lanthanum partly inhibited the cellular activation by CALP2. We conclude that alveolar macrophages, but not neutrophils or eosinophils, can produce extremely high amounts of reactive oxygen species when stimulated via the calcium/CaM pathway. These results may contribute to new therapeutic strategies against oxidative stress in airway diseases.
Asunto(s)
Calmodulina/metabolismo , Proteínas Portadoras/farmacología , Macrófagos Alveolares/metabolismo , Péptidos/farmacología , Superóxidos/metabolismo , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Calcio/metabolismo , Agonistas de los Canales de Calcio/metabolismo , Calmodulina/antagonistas & inhibidores , Proteínas Portadoras/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Cobayas , Péptidos y Proteínas de Señalización Intercelular , Macrófagos Alveolares/efectos de los fármacos , Masculino , Compuestos Onio/farmacología , Péptidos/antagonistas & inhibidores , Sulfonamidas/farmacología , Superóxido Dismutasa/farmacologíaRESUMEN
We previously described two novel peptides, Ca2+-like peptide (CALP) 1 and CALP2, which interact with Ca2+-binding EF hand motifs, and therefore have the characteristics to define the role of the Ca2+-sensing regulatory protein calmodulin in asthma. In the present study, the effects of the calcium-like peptides were investigated in an animal model for allergic asthma. For that purpose, sensitized guinea pigs were intratracheally pretreated with CALP1 or CALP2. Thirty minutes later, the animals were challenged with aerosolized ovalbumin. Acute bronchoconstriction was measured as well as characteristic features of asthma 6 and 24 hours (h) after challenge. Neither CALP1 nor CALP2 prevented the anaphylactic response elicited by ovalbumin challenge. However, CALP1 pretreatment attenuated the influx of inflammatory cells in the lungs 6 h after challenge. Furthermore, radical production by these cells was diminished both 6 and 24 h after challenge. Moreover, CALP1 completely inhibited airway hyperresponsiveness in vitro 24 h after challenge. We conclude that CALP1, as a selective calmodulin agonist, inhibits the development of asthmatic features probably via the attenuation of mast cell degranulation and radical production. Specific modulation of calmodulin activity might therefore be a potential new target for the treatment of allergic asthma.