Custom-made Microdialysis Probe Design

Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand to monitor physiological, ...

Goethe University of Frankfurt

Detergents are frequently used for the solubilization of membrane proteins during and after purification steps. Unfortunately some of these detergents impair chromatographic separations and mass spectrometry (MS) analysis. Perfusion reversed-phase high-performance liquid chromatography (RP-HPLC) using POROS materials is suited for separating intact proteins solubilized by detergents due to the particles' highly diffusive pores and chemical stability. In this article, the use of perfusive reversed-phase material packed into small inner diameter capillary columns is presented as a cheap, rapid, and efficient method for the removal of different types of detergents from protein solutions. The ability to purify and separate the subunits of membrane protein complexes with self-packed capillary columns is exemplified for bovine cytochrome bc(1) complex. Even highly hydrophobic subunits can be detected in collected fractions by intact mass measurements and identified after proteolytic digestion and matrix-assisted laser desorption/ionization tandem MS (MALDI MS/MS). The comparison with a gel-based approach shows that this method is a valuable alternative for purification and separation of intact proteins with subsequent MS analysis and that hydrophobic proteins are even better represented in the LC-based approach.

Perfusion reversed-phase high-performance liquid chromatography for protein separation from detergent-containing solutions: an alternative to gel-based approaches.

D&eacute;tergents sont fr&eacute;quemment utilis&eacute;s pour la solubilisation des prot&eacute;ines membranaires pendant et apr&egrave;s les &eacute;tapes de purification. Malheureusement, certains de ces d&eacute;tergents nuire s&eacute;parations chromatographiques et analyse de spectrom&eacute;trie de masse (MS). Chromatographie liquide haute performance en phase invers&eacute;e &agrave; perfusion (RP-HPLC) en utilisant des mat&eacute;riaux POROS est adapt&eacute; pour la s&eacute;paration des prot&eacute;ines intactes solubilis&eacute;s par les d&eacute;tergents en raison des particules hautement diffusives pores et stabilit&eacute; chimique. Dans cet article, l&#39;utilisation du mat&eacute;riel de phase invers&eacute;e perfusive emball&eacute; dans les colonnes capillaires de petit diam&egrave;tre int&eacute;rieur est pr&eacute;sent&eacute;e comme une m&eacute;thode bon march&eacute;e, rapide et efficace pour l&#39;&eacute;limination de diff&eacute;rents types de d&eacute;tergents des solutions prot&eacute;iques. La capacit&eacute; de purifier et de s&eacute;parer les sous-unit&eacute;s des complexes de prot&eacute;ine de membrane avec des colonnes capillaires self-packed est illustr&eacute;e pour cytochrome bovine non-maths complexes. Sous-unit&eacute;s m&ecirc;me fortement hydrophobes peuvent &ecirc;tre d&eacute;tect&eacute;es dans les fractions collect&eacute;es par des mesures de masse intacts et identifi&eacute;es apr&egrave;s digestion prot&eacute;olytique et tandem de d&eacute;sorption-ionisation laser assist&eacute;e par matrice (MALDI-MS/MS) de MS. La comparaison avec une approche ax&eacute;e sur le gel montre que cette m&eacute;thode est une alternative int&eacute;ressante pour la purification et la s&eacute;paration des prot&eacute;ines intactes avec analyse subs&eacute;quente de MS, et que les prot&eacute;ines hydrophobes sont encore mieux repr&eacute;sent&eacute; dans l&#39;approche ax&eacute;e sur la LC.

Chromatographie liquide &agrave; haute performance en phase invers&eacute;e perfusion pour s&eacute;paration de prot&eacute;ines de d&eacute;tergent contenant des solutions : une alternative aux approches ax&eacute;es sur le gel.

Detergenti sono frequentemente utilizzate per la solubilizzazione delle proteine di membrana durante e dopo la procedura di purificazione. Purtroppo alcuni di questi detergenti compromettere le separazioni cromatografiche e analisi di spettrometria di massa (MS). Cromatografia liquida in perfusione controfase ad alte prestazioni (RP-HPLC) utilizzando materiali POROS &egrave; adatto per la separazione di proteine intatte solubilizzate da detergenti a causa delle particelle altamente diffusivi pori e stabilit&agrave; chimica. In questo articolo, l&#39;uso di materiale di controfase perfusive confezionato in colonne capillari di piccolo diametro interno viene presentato come un metodo economico, rapido ed efficace per la rimozione di diversi tipi di detersivi da soluzioni di proteine. La capacit&agrave; di purificare e separare le subunit&agrave; dei complessi della proteina di membrana con le colonne capillari self-packed &egrave; esemplificata per bovini citocromo bc(1) complesso. Anche altamente idrofobica subunit&agrave; pu&ograve; essere rilevata in raccolta differenziata da misurazioni di massa intatte e identificate dopo la digestione proteolitica e tandem di desorbimento/ionizzazione laser assistita da matrice (MALDI MS/MS) MS. Il confronto con un approccio basato su gel dimostra che questo metodo &egrave; una valida alternativa per la purificazione e separazione delle proteine intatte con successive analisi MS e che le proteine idrofobe sono anche meglio rappresentati nell&#39;approccio basato su LC.

Perfusione controfase high-performance liquid chromatography per la separazione di proteine da detergenti contenenti soluzioni: un&#39;alternativa ad approcci basati su gel.

Graphite as MALDI matrix or in combination with other substances has been reported in recent years. Here, we demonstrate that graphite can be used as target coating supporting the crystallization of the Î±-cyano-4-hydroxycinnamic acid matrix. A conventional dried-droplet preparation of matrix and analyte solution on a graphite-coated metal target leads to a thin, uniform layer of cubic crystals with about 1 Î¼m edge length. Commercially available graphite powder of 1-2 Î¼m particle size is gently wiped over the target using a cotton Q-tip, leading to an ultra-thin, not-visible film. This surface modification considerably improves analysis of peptides and proteins for MALDI MS using conventional dried-droplet preparation. Compared with untreated targets, the signal intensities of standard peptides are up to eight times higher when using the graphite supported crystallization. The relative standard deviation in peak area of angiotensin II for sample amounts between 1 and 50 fmol is reduced to about 15 % compared with 45 % for untreated sample holders. For a quantification of 1 fmol of the peptide using an internal standard the coefficient of variation is reduced to 3.5 % from 8 %. The new graphite supported preparation (GSP) protocol is very simple and does not require any technical nor manual skills. All standard solvents for peptides and proteins can be used.

Graphite supported preparation (GSP) of Î±-cyano-4-hydroxycinnamic acid (CHCA) for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for peptides and proteins.

Graphite comme matrice MALDI ou en combinaison avec d&#39;autres substances a &eacute;t&eacute; signal&eacute; ces derni&egrave;res ann&eacute;es. Ici, nous d&eacute;montrons que le graphite peut &ecirc;tre utilis&eacute; comme cible de rev&ecirc;tement soutenant la cristallisation de la matrice d&#39;acide &alpha;-cyano-4-hydroxycinnamique. Une pr&eacute;paration de gouttelettes s&eacute;ch&eacute;es classique de solution matrix et analyte sur une cible m&eacute;tallique graphite rev&ecirc;tu m&egrave;ne &agrave; une mince couche uniforme de cristaux cubiques avec environ 1 &mu;m de long d&#39;affilage. Poudre de graphite disponible dans le commerce de 1-2 &mu;m granulom&eacute;trie est essuy&eacute; doucement au-dessus de la cible &agrave; l&#39;aide d&#39;un coton de q-Tip, conduisant &agrave; un film ultra-mince, n&#39;est pas visible. Cette modification de surface am&eacute;liore consid&eacute;rablement l&#39;analyse des peptides et des prot&eacute;ines pour MALDI MS en utilisant classique gouttelettes s&eacute;ch&eacute;es pr&eacute;paration. Par rapport aux cibles non trait&eacute;es, les intensit&eacute;s du signal de peptides standards sont jusqu&#39;&agrave; huit fois plus &eacute;lev&eacute;es lorsque l&#39;utilisation du graphite prise en charge de cristallisation. L&#39;&eacute;cart type relatif &agrave; la surface du pic de l&#39;angiotensine II pour les quantit&eacute;s d&#39;&eacute;chantillon entre 1 et 50 fmol est r&eacute;duite &agrave; environ 15 %, contre 45 % pour les titulaires de l&#39;&eacute;chantillon non trait&eacute;. Pour un dosage de 1 fmol du peptide &agrave; l&#39;aide d&#39;un &eacute;talon interne, le coefficient de variation est r&eacute;duit &agrave; 3,5 % de 8 %. Le nouveau protocole de pr&eacute;paration (GSP) de graphite pris en charge est tr&egrave;s simple et ne n&eacute;cessite pas des comp&eacute;tences techniques ni manuels. Tous les solvants standards pour les peptides et les prot&eacute;ines peuvent &ecirc;tre utilis&eacute;s.

Graphite soutenu pr&eacute;paration (SPG) de l&#39;acide &alpha;-cyano-4-hydroxycinnamique (ACSSD) pour spectrom&eacute;trie masse d&eacute;sorption-ionisation laser assist&eacute;e par matrice (MALDI-MS) aux prot&eacute;ines et peptides.

Grafite come matrice MALDI o in combinazione con altre sostanze &egrave; stato segnalato negli ultimi anni. Qui, ci dimostrano che la grafite pu&ograve; essere usato come bersaglio il rivestimento sostenendo la cristallizzazione della matrice acida &alpha;-ciano-4-idrossicinnamici. Una preparazione convenzionale droplet essiccati di matrice e analita soluzione su un bersaglio di metallo rivestito in grafite conduce ad uno strato sottile e uniforme di cristalli cubici con lunghezza del bordo circa 1 &mu;m. Polvere di grafite commercialmente disponibile di dimensione delle particelle di 1-2 &mu;m &egrave; asciug&ograve; delicatamente sopra la porta usando un cotone q-tip, portando a un film ultra-sottile, non visibile. Questa modificazione superficiale migliora considerevolmente analisi dei peptidi e delle proteine per MALDI MS utilizzando la preparazione convenzionale droplet essiccati. Rispetto agli obiettivi non trattati, l&#39;intensit&agrave; di segnale dei peptidi standard sono fino a otto volte superiore quando si utilizza la grafite supportato cristallizzazione. La deviazione standard relativa, nell&#39;area del picco dell&#39;angiotensina II per la quantit&agrave; di campione tra 1 e 50 fmol &egrave; ridotto a circa il 15% rispetto al 45% per i possessori di campione non trattato. Per una quantificazione di 1 fmol del peptide utilizzando uno standard interno il coefficiente di variazione &egrave; ridotta al 3,5% dall&#39;8%. Il nuovo protocollo di preparazione (SPG) grafite supportati &egrave; molto semplice e non richiede alcune abilit&agrave; tecniche n&eacute; manuale. Possono essere utilizzati tutti i solventi standard per peptidi e proteine.

Grafite supportato preparazione (GSP) di acido &alpha;-ciano-4-idrossicinnamici (CHCA) per matrix-assisted laser desorption/ionizzazione spettrometria di massa (MALDI-MS) per peptidi e proteine.

Although the extraction of intact proteins from polyacrylamide gels followed by mass spectrometric molecular mass determination has been shown to be efficient, there is room for alternative approaches. Our study evaluates ethylene glycol diacrylate, a cleavable cross-linking agent used for a new type of dissolvable gels. It attains an ester linkage that can be hydrolyzed in alkali conditions. The separation performance of the new gel system was tested by 1D and 2D SDS-PAGE using the outer chloroplast envelope of Pisum sativum as well as a soluble protein fraction of human lymphocytes, respectively. Gel spot staining (CBB), dissolving, and extracting were conducted using a custom-developed workflow. It includes protein extraction with an ammonia-SDS buffer followed by methanol treatment to remove acrylamide filaments. Necessary purification for MALDI-TOF analysis was implemented using methanol-chloroform precipitation and perfusion HPLC. Both cleaning procedures were applied to several standard proteins of different molecular weight as well as 'real' biological samples (8-75 kDa). The protein amounts, which had to be loaded on the gel to detect a peak in MALDI-TOF MS, were in the range of 0.1 to 5 μg, and the required amount increased with increasing mass.

MALDI analysis of proteins after extraction from dissolvable ethylene glycol diacrylate cross-linked polyacrylamide gels.

Microdialysis is an established technique for collecting small molecular weight substances (e.g. neurotransmitter and energy metabolites) from the extracellular space. The major element of microdialysis is the probe which contains a semi-permeable membrane and is exposed to the interstitial space. As the microdialysis technique has major advantages, e.g. versatility and use in awake animals, commercially produced probes are in great demand.

Jan Gorka

Department of Pharmaceutical Chemistry

Goethe University of Frankfurt

Custom-made Microdialysis Probe Design

Jan Gorka

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pharmaceutical Chemistry

Goethe University of Frankfurt

Custom-made Microdialysis Probe Design

Department of Pharmaceutical Chemistry