proteome-wide-quantification-labeling-homogeneity-at-single-molecule

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

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Poly( L-lysine) (PLL)/hyaluronic acid (HA) multilayers are films whose thickness increases exponentially with the number of deposition steps. Such a growth process was attributed to the diffusion, in and out of the whole film, of at least one of the polyelectrolytes constituting the film. In the case of PLL/HA, PLL is known to be the diffusing species. In order to better understand the growth mechanism of such films, it is of primary importance to well characterize the diffusion process of the polyelectrolytes in the multilayer. This process is studied here by fluorescence recovery after pattern photobleaching. We show that the diffusion behavior is different when we consider either PLL chains that are deposited on top of the film or PLL chains embedded in the film, even below only one HA layer. For chains that are embedded, we find two populations: a mobile one with a diffusion coefficient, D, of the order of 0.1 microm(2) x s(-1) and a population that appears immobile ( D < 0.001 microm(2) x s(-1)). For chains deposited on top of the multilayer, a third population appears which is rapidly diffusing ( D congruent with 1 microm(2) x s(-1)). These results confirm the validity of the model generally accepted for the exponential growth process and in particular the existence of up to three subgroups of PLL chains from the point of view of their diffusion coefficient.

Dynamics of poly(L-lysine) in hyaluronic acid/poly(L-lysine) multilayer films studied by fluorescence recovery after pattern photobleaching.

Poly (L-lysine) (PLL) / multicouches de l&#39;acide hyaluronique (HA) sont des films dont l&#39;&eacute;paisseur augmente exponentiellement avec le nombre d&#39;&eacute;tapes de d&eacute;p&ocirc;ts. Un tel processus de croissance &eacute;tait attribuable &agrave; la diffusion, dans et hors de tout le film, d&#39;au moins un des poly&eacute;lectrolytes constituant le film. Dans le cas de la PLL/HA, PLL est connue pour &ecirc;tre l&#39;esp&egrave;ce de diffusion. Afin de mieux comprendre le m&eacute;canisme de croissance de ces films, il est primordial de bien caract&eacute;riser le processus de diffusion des poly&eacute;lectrolytes dans le multicouche. Ce processus est &eacute;tudi&eacute; ici par r&eacute;cup&eacute;ration de fluorescence apr&egrave;s photoblanchiment de mod&egrave;le. Nous montrons que le comportement de diffusion est diff&eacute;rent si l&#39;on consid&egrave;re les cha&icirc;nes PLL qui sont d&eacute;pos&eacute;s sur le dessus du film ou cha&icirc;nes PLL int&eacute;gr&eacute;es dans le film, m&ecirc;me en dessous de HA une seule couche. Pour les cha&icirc;nes qui sont int&eacute;gr&eacute;s, on trouve deux populations : un mobile avec un coefficient de diffusion, D, de l&#39;ordre de 0,1 microm(2) x s(-1) et une population qui semble immobile (D &lt; 0,001 microm(2) x s(-1)). Pour les cha&icirc;nes d&eacute;pos&eacute;s sur le dessus de la multicouche, une troisi&egrave;me population appara&icirc;t qui est diffuser rapidement (D congruente avec 1 microm(2) x s(-1)). Ces r&eacute;sultats confirment la validit&eacute; du mod&egrave;le g&eacute;n&eacute;ralement admises pour le processus de croissance exponentielle, et en particulier l&#39;existence de trois sous-groupes de PLL cha&icirc;nes du point de vue de leur coefficient de diffusion.

Dynamique des poly(L-lysine) dans hyaluronique acid/poly(L-lysine) films multicouches &eacute;tudi&eacute;s par r&eacute;cup&eacute;ration de fluorescence apr&egrave;s photoblanchiment de mod&egrave;le.

Poli (L-lisina) (PLL) / multistrati di acido ialuronico (HA) sono film di cui spessore aumenta esponenzialmente con il numero di passaggi di deposizione. Un tale processo di crescita &egrave; stato attribuito alla diffusione, dentro e fuori tutto il film, almeno uno dei polielettroliti che costituiscono il film. Nel caso del PLL/HA, PLL &egrave; conosciuto per essere la specie diffondente. Per capire meglio il meccanismo di crescita di tali film, &egrave; di primaria importanza per caratterizzare bene il processo di diffusione del polielettrolita nel multistrato. Questo processo &egrave; studiato qui da recupero di fluorescenza dopo modello photobleaching. Dimostriamo che il comportamento di diffusione &egrave; diverso quando consideriamo catene PLL che sono depositate sopra il film o catene PLL incorporate nel film, anche di sotto di un solo strato HA. Per le catene che sono incorporate, troviamo due popolazioni: una mobile con un coefficiente di diffusione, D, dell&#39;ordine di 0.1 microm(2) x s(-1) e una popolazione che appare immobile (D &lt; 0.001 microm(2) x s(-1)). Per catene depositate sopra il multistrato, una terza popolazione appare che &egrave; rapidamente diffondendo (D congruenti con 1 microm(2) x s(-1)). Questi risultati confermano la validit&agrave; del modello generalmente accettato per il processo di crescita esponenziale e in particolare l&#39;esistenza di tre sottogruppi di PLL catene dal punto di vista del loro coefficiente di diffusione.

Dinamica di poly(L-lysine) nel film multistrato di ialuronico acid/poly(L-lysine) studiato dal recupero della fluorescenza dopo modello photobleaching.

Millions of teeth are saved each year by root canal therapy. Although current treatment modalities offer high levels of success for many conditions, an ideal form of therapy might consist of regenerative approaches in which diseased or necrotic pulp tissues are removed and replaced with healthy pulp tissue to revitalize teeth. Melanocortin peptides (alpha-MSH) possess anti-inflammatory properties in many acute and chronic inflammatory models. Our recent studies have shown that alpha-MSH covalently coupled to poly-l-glutamic acid (PGA-alpha-MSH) retains anti-inflammatory properties on rat monocytes. This study aimed to define the effects of PGA-alpha-MSH on dental pulp fibroblasts. Lipopolysaccharide (LPS)-stimulated fibroblasts incubated with PGA-alpha-MSH showed an early time-dependent inhibition of TNF-alpha, a late induction of IL-10, and no effect on IL-8 secretion. However, in the absence of LPS, PGA-alpha-MSH induced IL-8 secretion and proliferation of pulp fibroblasts, whereas free alpha-MSH inhibited this proliferation. Thus, PGA-alpha-MSH has potential effects in promoting human pulp fibroblast adhesion and cell proliferation. It can also reduce the inflammatory state of LPS-stimulated pulp fibroblasts observed in gram-negative bacterial infections. These effects suggest a novel use of PGA-alpha-MSH as an anti-inflammatory agent in the treatment of endodontic lesions. To better understand these results, we have also used the multilayered polyelectrolyte films as a reservoir for PGA-alpha-MSH by using not only PLL (poly-l-lysine) but also the Dendri Graft poly-l-lysines (DGL(G4)) to be able to adsorb more PGA-alpha-MSH. Our results indicated clearly that, by using PGA-alpha-MSH, we increase not only the viability of cells but also the proliferation. We have also analyzed at the nanoscale by atomic force microscopy these nanostructured architectures and shown an increase of thickness and roughness in the presence of PGA-alpha-MSH incorporated into the multilayered film (PLL-PGA-alpha-MSH)(10) or (DGL(G4)-PGA-alpha-MSH)(10) in accordance with the increase of the proliferation of the cells growing on the surface of these architectures. We report here the first use of nanostructured and functionalized multilayered films containing alpha-MSH as a new active biomaterial for endodontic regeneration.

Nanostructured assemblies for dental application.

Fluorescence-based electrophoresis has been widely used for proteome analysis in which every protein species in cells is labeled with a fluorescent dye, separated by electric migration, and quantified using fluorescence detection. The ultimate limit of sensitivity for this approach could be reached by single-molecule fluorescence imaging and counting individual proteins, requiring exhaustive fluorescent labeling of proteins across molecular populations and species. However, it remains unclear how homogeneous the fluorescence labeling of individual protein molecules of each species is across the proteome. To address this question, we developed a method to measure the labeling homogeneity based on a single-molecule fluorescence counting assay. Our results reveal that the proportion of proteins labeled with at least one dye, called labeling occupancy (LO), was 35% for fluorescently labeled BSA using existing protocols. We then found that the LO could be improved to 82% under high pH and surfactant-rich conditions. Furthermore, when a proteome sample from a human cell lysate was analyzed, the total LO was 71%, whereby the values varied between 50 and 90% for low and high molecular weight proteome fractions, respectively. The results support the possibility of sensitive detection of proteins using single-molecule counting with fluorescent labeling at the proteome scale.

Youri Arntz

Laboratoire de Biomatériaux et Bioimagerie

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Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level