We have investigated six nanomaterials for their applicability as surfaces for the analyses of peptides and proteins using surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). Gold nanoparticles (NPs) were useful nanomaterials for small analytes (e.g., glutathione); Pt nanosponges and Fe 3 O 4 NPs were efficient nanomaterials for proteins, with an upper detectable mass limit of ca. 25 kDa. Nanomaterials have several advantages over organic matrices, including lower limits of detection for small analytes and lower batch-to-batch variations (fewer problems associated with "sweet spots"), when used in laser desorption/ionization mass spectrometry. (J Am Soc Mass Spectrom 2010, 21, 1204 -1207) © 2010 American Society for Mass Spectrometry S urface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) was developed recently using nanomaterials rather than organic compounds as matrices, for the determination of analytes of interest. For example, laser desorption/ionization (LDI) of intact proteins and protein aggregates in the presence of glycerol has been demonstrated using cobalt particles (ca. 30 nm) [1]. Similar to the role played by organic matrices, the particles absorb energy from the laser irradiation and transfer it efficiently to the analytes, thereby inducing desorption and ionization. Mixtures of graphite particles (2-150 m) and glycerol have been employed in the analysis of proteins and peptides [2,3]. Several other nanomaterials, including carbon nanotubes, nanodiamonds, and various nanoparticles (NPs, namely SiO 2 , ZnS, TiO 2 , Fe 3 O 4 , Fe 3 O 4 /TiO 2 , and Au) are also useful-without the addition of glycerol-for SALDI-MS [4 -12]. Because of their unique chemical and physical properties, NPs can also act as selective probes and/or efficient ionization nanomaterials. For example, Au and TiO 2 NPs are suitable for the concentration and ionization of aminothiols and catechins, respectively, in SALDI-MS [8,11]. One other advantage of using NPs is that fewer "sweet spots" are formed, thereby maximizing reproducibility. Although NPs have been used successfully for the determination of a range of analytes (from small analytes to proteins), a review of the literature reveals that the various NPs provide quite different results in terms of sensitivity, reproducibility, and mass range. Thus, our aim in this study was to evaluate the performance of several types of NPs for the analysis of peptides and proteins. ExperimentalSix nanomaterials-Au NPs, TiO 2 NPs, Se NPs, CdTe quantum dots (QDs), Fe 3 O 4 NPs, and Pt nanosponges (NSPs)-were tested for the SALDI-MS-based analyses of peptides and proteins; they were prepared in aqueous solutions and characterized according to procedures described in the literature [8,11,[13][14][15][16]. A twolayer preparation method was applied to deposit the nanomaterials and samples onto the metal plates used in SALDI-MS. First, one of the nanomaterial solutions (1 L) was deposited into one of the wells of the MS plate and dried under ambient cond...
We have analyzed peptides, proteins, and protein-drug complexes through surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) using HgTe nanostructures as matrixes. We investigated the effects of several parameters, including the concentration of the HgTe nanostructures, the pH of the buffer, and the concentration of salt, on the performance of this system. When HgTe nanostructures are used as matrixes, [M + H](+) ions were the dominant signals. Relative to other commonly used nanomaterials, HgTe nanostructures provided lower background signals from metal clusters, fewer fragment ions, less interference from alkali-adducted analyte ions, and a higher mass range (up to 150,000 Da). The present approach provides limits of detection for angiotensin I and bovine serum albumin of 200 pM and 14 nM, respectively, with great reproducibility (RSD: <25%). We validated the applicability of this method through the detections of (i) the recombinant proteins that were transformed in E. coli, (ii) the specific complex between bovine serum albumin and l-tryptophan, and (iii) a carbonic anhydrase-acetazolamide complex. Our results suggest that this novel and simple SALDI-MS approach using HgTe nanostructures as matrixes might open several new ways for proteomics and the analysis of drug-protein complexes.
Daily rhythms in behavior and physiology are coordinated by an endogenous clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. This central pacemaker also relays day length information to allow for seasonal adaptation, a process for which melatonin signaling is essential. How the SCN encodes day length is not fully understood. MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression by directing target mRNAs for degradation or translational repression. The miR-132/212 cluster plays a key role in facilitating neuronal plasticity, and miR-132 has been shown previously to modulate resetting of the central clock. A recent study from our group showed that miR-132/212 in mice is required for optimal adaptation to seasons and non-24-hour light/dark cycles through regulation of its target gene, methyl CpG-binding protein (MeCP2), in the SCN and dendritic spine density of SCN neurons. Furthermore, in the seasonal rodent Mesocricetus auratus (Syrian hamster), adaptation to short photoperiods is accompanied by structural plasticity in the SCN independently of melatonin signaling, thus further supporting a key role for SCN structural and, in turn, functional plasticity in the coding of day length. In this commentary, we discuss our recent findings in context of what is known about day length encoding by the SCN, and propose future directions.
This manuscript describes dynamic coating of capillaries with poly(L-lysine) (PLL) and silica nanoparticles (SiO 2 NPs) and use of the as-prepared capillaries for the separation of biogenic amines and acids by CE in conjunction with LIF detection. The directions of EOF are controlled by varying the outmost layer of the capillaries with PLL and SiO 2 NPs, respectively. Over the pH range 3.0-5.0, the (PLL-SiO 2 NP) n -PLL capillaries have an EOF toward the anodic end and are more suitable for the separation of acids with respect to speed, while the (PLL-SiO 2 NP) n capillaries have an EOF toward the cathodic end and are more suitable for the separation of biogenic amines regarding speed and sensitivity. The separations of standard solutions containing five amines and two acids by CE with LIF detection using (PLL-SiO 2 NP) 2 -PLL and (PLL-SiO 2 NP) 3 capillaries were accomplished within 10 and 7 min, providing plate numbers of 3.8 and 5.0610 4 plates/m for 5-hydroxytryptamine (5-HT), respectively. The LODs for 5-HT and 5-hydroxyindole-3-acetic acid (5-HIAA) are 32 and 2 nM and 0.2 and 1.5 nM when using the (PLL-SiO 2 NP) 2 -PLL and (PLL-SiO 2 NP) 3 capillaries, respectively. Identification and quantification of 5-HIAA, homovanillic acid, and DL-vanillomandelic acid in urine samples from a male before and after drinking green tea were tested to validate practicality of the present approach. The results show that the (PLLSiO 2 NP) 2 -PLL capillary provides greater resolving power, while the (PLL-SiO 2 NP) 3 capillary provides better sensitivity, higher efficiency, and longer durability for the separation of the amines and acids.
One of the major biological functions of the retinal pigmented epithelium (RPE) is the clearance of shed photoreceptor outer segments (POS) through a multistep process resembling phagocytosis. RPE phagocytosis helps maintain the viability of photoreceptors that otherwise could succumb to the high metabolic flux and photo-oxidative stress associated with visual processing. The regulatory mechanisms underlying phagocytosis in the RPE are not fully understood, although dysfunction of this process contributes to the pathogenesis of multiple human retinal degenerative disorders, including age-related macular degeneration. Here, we present an integrated transcriptomic, proteomic, and phosphoproteomic analysis of phagocytosing RPE cells, utilizing three different experimental models: the human-derived RPE-like cell line ARPE-19, cultured murine primary RPE cells, and RPE samples from live mice. Our combined results indicated that early stages of phagocytosis in the RPE are mainly characterized by pronounced changes in the protein phosphorylation level. Global phosphoprotein enrichment analysis revealed involvement of PI3K/Akt, mechanistic target of rapamycin (mTOR), and MEK/ERK pathways in the regulation of RPE phagocytosis, confirmed by immunoblot analyses and phagocytosis assays. Most strikingly, phagocytosis of POS by cultured RPE cells was almost completely blocked by pharmacological inhibition of phosphorylation of Akt. Our findings, along with those of previous studies, indicate that these phosphorylation events allow the RPE to integrate multiple signals instigated by shed POS at different stages of the phagocytic process.
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