A novel two-laser interface for coupling capillary electrochromatography with ion-trap time-of-flight mass spectrometry

An interface has been developed for the hyphenation of capillary electrochromatography (CEC) with mass spectrometry (MS). Chromatographic eluate vaporization and selective analyte ionization occur within a quadrupole ion-trap, which permits significant instrument simplification when compared with the atmospheric pressure interfaces typically used for CEC-MS. Vaporization is achieved using laser desorption at 1064 nm while ionization is accomplished through UV photoionization. This two-step approach, through ionization laser wavelength selection, can provide ultratrace analysis with high selectivity. The mass spectrometer is a hybrid ion-trap time-of-flight (TOF) instrument in which the ion-trap is used in radio frequency-only mode, with DC-pulse ejection, to provide decoupling of the different timescales required for CEC separation and TOF mass analysis. The ion-trap is capable of accumulating ions over multiple laser shots. The mass resolution of the demonstration instrument was circa 1500. Preliminary CEC-MS runs have been recorded for mixtures containing polycyclic aromatic hydrocarbons. A concentration detection limit of 500 nM, for naphthalene in acetonitrile, has been determined for the interface

Lactate, a product of glycolytic metabolism, inhibits histone deacetylase activity and promotes changes in gene expression

Chemical inhibitors of histone deacetylase (HDAC) activity are used as experimental tools to induce histone hyperacetylation and deregulate gene transcription, but it is not known whether the inhibition of HDACs plays any part in the normal physiological regulation of transcription. Using both in vitro and in vivo assays, we show that lactate, which accumulates when glycolysis exceeds the cell’s aerobic metabolic capacity, is an endogenous HDAC inhibitor, deregulating transcription in an HDAC-dependent manner. Lactate is a relatively weak inhibitor (IC(50) 40 mM) compared to the established inhibitors trichostatin A and butyrate, but the genes deregulated overlap significantly with those affected by low concentrations of the more potent inhibitors. HDAC inhibition causes significant up and downregulation of genes, but genes that are associated with HDAC proteins are more likely to be upregulated and less likely to be downregulated than would be expected. Our results suggest that the primary effect of HDAC inhibition by endogenous short-chain fatty acids like lactate is to promote gene expression at genes associated with HDAC proteins. Therefore, we propose that lactate may be an important transcriptional regulator, linking the metabolic state of the cell to gene transcription

Determination of protein thiol reduction potential by isotope labeling and intact mass measurement

Oxidation/reduction of thiol residues in proteins is an important type of post-translational modification that is implicated in regulating a range of biological processes. The nature of the modification makes it possible to define a quantifiable electrochemical potential, E⊕, for oxidation/reduction that allows cysteine-containing proteins to be ranked based on their propensity to be oxidized. Measuring oxidation of cysteine residues in proteins is difficult using standard electrochemical methods but recently top-down mass-spectrometry has been shown to enable the quantification of E⊕ for thiol oxidations. In this paper we demonstrate that mass spectrometry of intact proteins can be used in combination with an isotopic labeling strategy and an automated data analysis algorithm to measure E⊕ for the thiols in both E Coli Thioredoxin 1 and Human Thioredoxin 1. Our methodology relies on accurate mass measurement of proteins using LC-MS analyses and does not necessarily require top-down fragmentation. As well as analyzing homogeneous protein samples, we also demonstrate that our methodology can be used to determine thiol E⊕ measurements in samples which contain mixtures of proteins. Thus the combination of experiential methodology and data analysis regime have the potential to make such measurements in a high-throughput manner and in a manner more accessible to a broad community of protein scientists

The chemical basis of serine palmitoyltransferase inhibition by myriocin

Sphingolipids (SLs) are essential components of cellular membranes formed from the condensation of L-serine and a long-chain acyl thioester. This first step is catalyzed by the pyridoxal-5'-phosphate (PLP)-dependent, enzyme serine palmitoyltransferase (SPT) which is a promising therapeutic target. The fungal natural product myriocin is a potent inhibitor of SPT and is widely used to block SL biosynthesis despite a lack of a detailed understanding of its molecular-mechanism. By combining spectroscopy, mass spectrometry, X-ray crystallography, and kinetics, we have characterized the molecular details of SPT inhibition by myriocin. Myriocin initially forms an external aldimine with PLP at the active site, and a structure of the resulting co-complex explains its nanomolar affinity for the enzyme. This co-complex then catalytically degrades via an unexpected 'retro-aldol-like' cleavage mechanism to a C18 aldehyde which in turn acts as a suicide inhibitor of SPT by covalent modification of the essential catalytic lysine. This surprising dual mechanism of inhibition rationalizes the extraordinary potency and longevity of myriocin inhibition.</p

Microscale Volcano of Microdroplet by ZnO Surface Acoustic Waves

Microvolcano like surface acoustic wave (SAW) nebulization/ atomization has been realised on ZnO film based SAW devices. The SAW nebulization (SAWN) process has been observed to produce significant mist generation and ejected satellite droplets. By modifying the geometry of the interdigitated transducers to reduce the wavelengths from 400 μm to 64 μm, higher frequency SAW nebulization has been achieved by increasing radio frequency (RF) driving frequencies from 11.8 MHz to 64 MHz respectively

Nebulization of water/glycerol droplets generated by ZnO/Si surface acoustic wave devices

Efficient nebulization of liquid sessile droplets (water and water/glycerol mixtures) was investigated using standing waves generated using ZnO/Si surface acoustic wave (SAW) devices under different RF powers, frequencies and liquid viscosity (varied glycol concentrations in water). At such high RF powers, there are strong competitions between vertical jetting and nebulization. At lower SAW frequencies of 12.3 and 23.37 MHz, significant capillary waves and large satellite droplets were generated before nebulization could be observed. At frequencies between 23.37 and 37.2 MHz, spreading, displacement or occasionally jetting of the parent sessile droplet was frequently observed before a significant nebulization occurred. When the SAW frequencies were increased from 44.44 to 63.3 MHz, the minimum RF power to initiate droplet nebulization was found to increase significantly, and jetting of the parent droplet before nebulization became significant, although the average size of the nebulized particles and ejected satellite droplets appeared to decrease with an increase in frequency. With the increase of glycerol concentration in the test sessile droplets (or increase in liquid viscosity), nebulization became difficult due to the increased SAW damping rate inside the liquid. Acoustic heating effects were characterized to be insignificant and did not show apparent contributions to the nebulization process due to silicon substrate’s natural effect as an effective heat sink and the employment of a metallic holder beneath the ZnO/Si SAW device substrates

Sensitive, specific and quantitative FT-ICR mass spectrometry of combinatorial post-translational modifications in intact histone H4

We describe a quantitative Fourier transform ion cyclotron resonance mass spectrometric (FTICR MS) analysis of the relative proportions of post-translational modification states (PTMs) of core histones in cultured cells and tissues. A novel preseparation process using a monolithic column interfaced to a 12 T FTICR MS equipped with electron capture dissociation (ECD) yields very high mass accuracy spectra, allowing direct assignment of the PTMs present in the dominant modification states of intact H4, resolving a well recognized ambiguity between trimethylation and acetylation states. By eliminating preseparation, we also obtain a highly quantitative analysis of the distribution of H4 PTMs. Rapid, extensive, and reversible effects on PTMs induced by a histone deacetylase inhibitor indicate that H4 and other core histones are accessible to modification throughout the chromatin, not just in regions of active transcription. These methods provide tools for analysis of the histone code and its role in chromatin function