It is a prevalent concept that, in line with the Wobble Hypothesis, those tRNAs having an adenosine in the first position of the anticodon become modified to an inosine at this position. Sequencing the cDNA derived from the gene coding for cytoplasmic tRNAArgACG from several higher plants as well as mass spectrometric analysis of the isoacceptor has revealed that for this kingdom an unmodified A in the wobble position of the anticodon is the rule rather than the exception. In vitro translation shows that in the plant system the absence of inosine in the wobble position of tRNAArg does not prevent decoding. This isoacceptor belongs to the class of tRNA that is imported from the cytoplasm into the mitochondria of higher plants. Previous studies on the mitochondrial tRNA pool have demonstrated the existence of tRNAArgICG in this organelle. In moss the mitochondrial encoded distinct tRNAArgACG isoacceptor possesses the I34 modification. The implication is that for mitochondrial protein biosynthesis A-to-I editing is necessary and occurs by a mitochondrion-specific deaminase after import of the unmodified nuclear encoded tRNAArgACG.
wobble hypothesis; inosine; tRNAArg; deaminase; decoding; plant; mitochondrial tRNA import
As a light-driven water-plastoquinone oxidoreductase, Photosystem II produces molecular oxygen as an enzymatic product. Additionally, under a variety of stress conditions, reactive oxygen species are produced at or near the active site for oxygen evolution. In this study, Fourier-transform ion cyclotron resonance mass spectrometry was used to identify oxidized amino acid residues located in several core Photosystem II proteins (D1, D2, CP43 and CP47) isolated from spinach Photosystem II membranes. While the majority of these oxidized residues (81%) are located on the oxygenated solvent-exposed surface of the complex, several residues on the CP43 protein (354E, 355T, 356M and 357R) which are in close proximity (<15 Å) to the Mn4CaO5 active site are also modified. These residues appear to be associated with putative oxygen/reactive oxygen species exit channel(s) in the photosystem. These results are discussed within the context of a number of computational studies which have identified putative oxygen channels within the photosystem.
A method for the selective detection and quantification of peptide:oligonucleotide heteroconjugates, such as those generated by protein:nucleic acid cross-links, using capillary reversed-phase high performance liquid chromatography (cap-RPHPLC) coupled with inductively coupled plasma mass spectrometry detection (ICPMS) is described. The selective detection of phosphorus as 31P+, the only natural isotope, in peptide-oligonucleotide heteroconjugates is enabled by the elemental detection capabilities of the ICPMS. Mobile phase conditions that allow separation of heteroconjugates while maintaining ICPMS compatibility were investigated. We found that trifluoroacetic acid (TFA) mobile phases, used in conventional peptide separations, and hexafluoroisopropanol/triethylamine (HFIP/TEA) mobile phases, used in conventional oligonucleotide separations, both are compatible with ICPMS and enable heteroconjugate separation. The TFA-based separations yielded limits of detection (LOD) of ~40 ppb phosphorus, which is nearly seven times lower than the LOD for HFIP/TEA-based separations. Using the TFA mobile phase, 1-2 pmol of a model heteroconjugate were routinely separated and detected by this optimized capLC-ICPMS method.
Inductively coupled plasma mass spectrometry; Protein-nucleic acid cross-links; Elemental phosphorus detection; Liquid chromatography-mass spectrometry; Quantitative analysis
Under a variety of stress conditions, Photosystem II produces reactive oxygen species on both the reducing and oxidizing sides of the photosystem. A number of different sites including the Mn4O5Ca cluster, P680, PheoD1, QA, QB and cytochrome b559 have been hypothesized to produce reactive oxygen species in the photosystem. In this communication using Fourier-transform ion cyclotron resonance mass spectrometry we have identified several residues on the D1 and D2 proteins from spinach which are oxidatively modified and in close proximity to QA (D1 residues 239F, 241Q, 242E and the D2 residues 238P, 239T, 242E and 247M) and PheoD1 (D1 residues 130E, 133L and 135F). These residues may be associated with reactive oxygen species exit pathways located on the reducing side of the photosystem, and their modification may indicate that both QA and PheoD1 are sources of reactive oxygen species on the reducing side of Photosystem II.
Archaeosine (G+) is found at position 15 of many archaeal tRNAs. In Euryarchaeota, the G+ precursor, 7-cyano-7-deazaguanine (preQ0), is inserted into tRNA by tRNA-guanine transglycosylase (arcTGT) before conversion into G+ by ARChaeosine Synthase (ArcS). However, many Crenarchaeota known to harbor G+ lack ArcS homologs. Using comparative genomics approaches, two families that could functionally replace ArcS in these organisms were identified: 1) GAT-QueC, a two-domain family with an N-terminal glutamine amidotransferase class-II domain fused to a domain homologous to QueC, the enzyme that produces preQ0; 2) QueF-like, a family homologous to the bacterial enzyme catalyzing the reduction of preQ0 to 7-aminomethyl-7-deazaguanine. Here we show that these two protein families are able to catalyze the formation of G+ in a heterologous system. Structure and sequence comparisons of crenarchaeal and euryarchaeal arcTGTs suggest the crenarchaeal enzymes have broader substrate specificity. These results led to a new model for the synthesis and salvage of G+ in Crenarchaeota.
The use of traditional capillary electrophoresis (CE) to detect weak binding complexes is problematic due to the fast-off rate resulting in the dissociation of the complex during the separation process. Additionally, proteins involved in binding interactions often nonspecifically stick to the bare-silica capillary walls, which further complicates the binding analysis. Microchip CE allows flexibly positioning the detector along the separation channel and conveniently adjusting the separation length. A short separation length plus a high electric field enables rapid separations thus reducing both the dissociation of the complex and the amount of protein loss due to nonspecific adsorption during the separation process. Thrombin and a selective thrombin-binding aptamer were used to demonstrate the capability of microchip CE for the study of relatively weak binding systems that have inherent limitations when using the migration shift method or other CE methods. The rapid separation of the thrombin-aptamer complex from the free aptamer was achieved in less than 10 s on a single-cross glass microchip with a relatively short detection length (1.0 cm) and a high electric field (670 V/cm). The dissociation constant was determined to be 43 nM, consistent with reported results. In addition, aptamer probes were used for the quantitation of standard thrombin samples by constructing a calibration curve, which showed good linearity over two orders of magnitude with a limit of detection for thrombin of 5 nM at a 3-fold signal-to-noise ratio.
Microchip; Affinity capillary electrophoresis; Aptamer; Binding study
Herein we report the topochemical modification of polymer surfaces with perfluorinated aromatic azides. The aryl azides, which have quaternary amine or aldehyde functional groups, were linked to the surface of the polymer by UV irradiation. The polymer substrates used in this study were cyclic olefin copolymer (COC) and poly(methylmethacrylate) (PMMA). These substrates were characterized before and after modification, using reflection-absorption infrared spectroscopy (RAIRS), sessile water contact angle measurements, and X-ray photoelectron spectroscopy (XPS). Analysis of the surface confirmed the presence of an aromatic groups with aldehyde or quaternary amine functionality. Enzyme immobilization and patterning onto polymer surfaces were studied using confocal microscopy. Enzymatic digests were carried out on modified probes manufactured from thermoplastic substrates and the resulting peptide analysis was completed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).
A method for the separation and detection of oligonucleotides utilizing hydrophilic interaction liquid chromatography (HILIC) with inductively coupled plasma mass spectrometry (ICPMS) is described. Polythymidilic acids of various lengths (10, 15, 20 and 30 nucleotides) were separated under gradient HILIC conditions. Selective detection of oligonucleotides was possible through monitoring m/z 47, corresponding to 31P16O+, using ICPMS. Oxygen was used as a reaction gas in the collision/reaction cell to produce PO+ by reacting with phosphorus in the gas phase, thereby effectively eliminating the interferences for phosphorus normally seen at m/z 31. Limits of detection (LODs) were determined to be 1.69 pmol, 1.21 pmol, 1.0 pmol and 0.55 pmol loaded on column for the 10, 15, 20 and 30-mer, respectively.
Elemental detection; DNA; RNA; nucleic acids; phosphorus speciation; LC-MS
Direct detection of pseudouridine (ψ), an isomer of uridine, in RNA is challenging. The most popular method requires chemical derivatization using N-cyclohexyl-N'-β-(4-methylmorpholinumethyl) carbodiimide p-tosylate (CMCT) followed by radiolabeled primer extension mediated by reverse transcriptase. More recently, mass spectrometry (MS)-based approaches for sequence placement of pseudouridine in RNA have been developed. Nearly all of these approaches, however, only yield qualitative information regarding the presence or absence of pseudouridine in a given RNA population. Here, we have extended a previously developed liquid chromatography tandem mass spectrometry (LC-MS/MS) method to enable both the qualitative and quantitative analysis of pseudouridine. Quantitative selected reaction monitoring (SRM) assays were developed using synthetic oligonucleotides, with or without pseudouridine, and the results yielded a linear relationship between the ion abundance of the pseudouridine-specific fragment ion and the amount of pseudouridine-containing oligonucleotide present in the original sample. Using this quantitative SRM assay, the extent of pseudouridine hypomodification in the conserved T-loop of tRNA isolated from two different Escherichia coli strains was established.
Pseudouridine quantification; tRNA; Mass spectrometry; Selected reaction monitoring; SRM; Detection of RNA modifications; Oligonucleotides
Equilibrium constants, such as the dissociation constant (Kd), are a key measurement of noncovalent interactions that are of importance to the proper functioning of molecules in living systems. Frontal analysis (FA) is a simple and accurate capillary electrophoresis (CE) method for the determination of Kd. Microchip CE coupled with laser-induced fluorescence (LIF) detection was used to determine Kd of protein-DNA interactions using the FA method. A model system of immunoglobulin E (IgE) and the IgE-binding aptamer was selected to demonstrate the capability of FA in microchip CE. Because the fluorescence emission was dependent on the dye migration velocity, the velocity of the free aptamer was adjusted to be the same as that of the aptamer-IgE complex by setting up individual separation voltage configurations for the free and bound aptamers. The ratio of the free and bound aptamers in the equilibrium mixture was directly measured from the heights of their plateaus detected at 1.0 cm from the intersection of the microchip, and no internal standard was needed. The Kd of the IgE-aptamer pair was determined as 6 ± 2 nM which is consistent with the reported results (8 nM).
Capillary electrophoresis; Dissociation constant; Frontal analysis; Laser-induced fluorescence; Microchip
An LC-MS method based on the use of high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS) for profiling oligonucleotides synthesis impurities is described.
Oligonucleotide phosphorothioatediesters (phosphorothioate oligonucleotides), in which one of the non-bridging oxygen atoms at each phosphorus center is replaced by a sulfur atom, are now one of the most popular oligonucleotide modifications due to their ease of chemical synthesis and advantageous pharmacokinetic properties. Despite significant progress in the solid-phase oligomerization chemistry used in the manufacturing of these oligonucleotides, multiple classes of low-level impurities always accompany synthetic oligonucleotides. Liquid chromatography-mass spectrometry has emerged as a powerful technique for the identification of these synthesis impurities. However, impurity profiling, where the entire complement of low-level synthetic impurities is identified in a single analysis, is more challenging. Here we present an LC-MS method based the use of high resolution-mass spectrometry, specifically Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS or FTMS). The optimal LC-FTMS conditions, including the stationary phase and mobile phases for the separation and identification of phosphorothioate oligonucleotides, were found. The characteristics of FTMS enable charge state determination from single m/z values of low-level impurities. Charge state information then enables more accurate modeling of the detected isotopic distribution for identification of the chemical composition of the detected impurity. Using this approach, a number of phosphorothioate impurities can be detected by LC-FTMS including failure sequences carrying 3′-terminal phosphate monoester and 3′-terminal phosphorothioate monoester, incomplete backbone sulfurization and desulfurization products, high molecular weight impurities, and chloral, isobutyryl, and N3 (2-cyanoethyl) adducts of the full length product. When compared with low resolution LC-MS, ~60% more impurities can be identified when charge state and isotopic distribution information is available and used for impurity profiling.
Synthetic oligonucleotides; phosphorothioatediesters; high resolution mass spectrometry; LC-FTMS; accurate mass measurement
Parallel separations using capillary electrophoresis on a multilane microchip with multiplexed laser induced fluorescence detection is demonstrated. The detection system was developed to simultaneously record data on all channels using an expanded laser beam for excitation, a camera lens to capture emission, and a CCD camera for detection. The detection system enables monitoring of each channel continuously and distinguishing individual lanes without significant crosstalk between adjacent lanes. Multiple analytes can be analyzed on parallel lanes within a single microchip in a single run, leading to increased sample throughput. The pKa determination of small molecule analytes is demonstrated with the multilane microchip.
The modified nucleosides N2-methylguanosine and N22-dimethylguanosine in transfer RNA occur at five positions in the D and anticodon arms, and at positions G6 and G7 in the acceptor stem. Trm1 and Trm11 enzymes are known to be responsible for several of the D/anticodon arm modifications, but methylases catalyzing post-transcriptional m2G synthesis in the acceptor stem are uncharacterized. Here, we report that the MJ0438 gene from Methanocaldococcus jannaschii encodes a novel S-adenosylmethionine-dependent methyltransferase, now identified as Trm14, which generates m2G at position 6 in tRNACys. The 381 amino acid Trm14 protein possesses a canonical RNA recognition THUMP domain at the amino terminus, followed by a γ-class Rossmann fold amino-methyltransferase catalytic domain featuring the signature NPPY active site motif. Trm14 is associated with cluster of orthologous groups (COG) 0116, and most closely resembles the m2G10 tRNA methylase Trm11. Phylogenetic analysis reveals a canonical archaeal/bacterial evolutionary separation with 20–30% sequence identities between the two branches, but it is likely that the detailed functions of COG 0116 enzymes differ between the archaeal and bacterial domains. In the archaeal branch, the protein is found exclusively in thermophiles. More distantly related Trm14 homologs were also identified in eukaryotes known to possess the m2G6 tRNA modification.
We have developed a method to screen for pseudouridines in complex mixtures of small RNAs using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS). First, the unfractionated crude mixture of tRNAs is digested to completion with an endoribonuclease, such as RNase T1, and the digestion products are examined using MALDI-MS. Individual RNAs are identified by their signature digestion products, which arise through the detection of unique mass values after nuclease digestion. Next, the endonuclease digest is derivatized using N-cyclohexyl-N’-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate (CMCT), which selectively modifies all pseudouridine, thiouridine and 2-methylthio-6-isopentenyladenosine nucleosides. MALDI-MS determination of the CMCT-derivatized endonuclease digest reveals the presence of pseudouridine through a 252 Da mass increase over the underivatized digest. Proof-of-concept experiments were conducted using a mixture of Escherichia coli transfer RNAs and endoribonucleases T1 and A. More than 80% of the expected pseudouridines from this mixture were detected using this screening approach, even on a unfractionated sample of tRNAs. This approach should be particularly useful in the identification of putative pseudouridine synthases through detection of their target RNAs and can provide insight into specific small RNAs that may contain pseudouridine.
CMC derivatization; Endonucleases; RNase T1; RNase A; Pseudouridine; 4-thiouridine; tRNA; MALDI-MS; transfer RNA; non-coding RNA
A method has been developed to identify oligonucleotide-peptide heteroconjugates by accurate mass measurements using mass spectrometry. The fractional mass (the decimal fraction mass value following the monoisotopic nominal mass) for peptides and oligonucleotides is different due to their differing molecular compositions. This property has been used to develop the general conditions necessary to differentiate peptides and oligonucleotides from oligonucleotide-peptide heteroconjugates. Peptides and oligonucleotides generated by the theoretical digestion of various proteins and nucleic acids were plotted as nominal mass versus fractional mass. Such plots reveal that three nucleotides cross-linked to a peptide produce enough change in the fractional mass to be recognized from non-crosslinked peptides at the same nominal mass. Experimentally, a cytochrome c digest was spiked with an oligonucleotide-peptide heteroconjugate and conditions for analyzing the sample using liquid chromatography-mass spectrometry were optimized. Upon analysis of this mixture, all detected masses were plotted on a fractional mass plot and the heteroconjugate could be readily distinguished from non-crosslinked peptides. The method developed here can be incorporated into a general proteomics-like scheme for identifying protein-nucleic acid cross-links, and this method is equally applicable to characterizing cross-links generated from protein-DNA and protein-RNA complexes.
Fractional mass; mass excess; mass defect; cross-linking; oligonucleotide-peptide heteroconjugate
There is a continuing drive in microfluidics to transfer microchip systems from the more expensive glass microchips to cheaper polymer microchips. Here, we investigate using polyelectrolyte multilayers (PEM) as a coating system for poly (methylmethacrylate) (PMMA) microchips to improve their functionality. The multilayer system was prepared by layer-on-layer depositon of poly (diallydimethylammonium) chloride (PDAD) and polystyrene sulfonate (PSS). Practical aspects of coating PMMA microchips were explored. The multilayer buildup process was monitored using EOF measurements, and the stability of the PEM was investigated. The performance of the PEM-PMMA microchip was compared to those of a standard glass microchip and a PEM-glass microchip in terms of electroosmotic flow and separating two fluorescent dyes. Several key findings in the development of the multilayer coating procedure for PMMA chips are also presented. It was found that, with careful preparation, a PEM-PMMA microchip can be prepared that has properties comparable - and in some cases superior - to those of a standard glass microchip.
Our understanding of the structural organization of ribosome assembly intermediates, in particular those intermediates that result from mis-folding leading to their eventual degradation within the cell, is limited due to the lack of methods available to characterize assembly intermediate structures. Because conventional structural approaches, such as NMR, X-ray crystallography and cryo-EM, are not ideally suited to characterize the structural organization of these flexible and sometimes heterogeneous assembly intermediates, we have set out to develop an approach combining limited proteolysis with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) that might be applicable to ribonucleoprotein complexes as large as the ribosome. This study focuses on the limited proteolysis behavior of appropriately assembled ribosome subunits. Isolated subunits were analyzed using limited proteolysis and MALDI-MS and the results were compared to previous data obtained from 70S ribosomes. Generally, ribosomal proteins were found to be more stable in 70S ribosomes than in their isolated subunits, consistent with a reduction in conformational flexibility upon subunit assembly. This approach demonstrates that limited proteolysis combined with MALDI-MS can reveal structural changes to ribosomes upon subunit assembly or disassembly, and provides the appropriate benchmark data from 30S, 50S and 70S proteins to enable studies of ribosome assembly intermediates.
MALDI-MS; 30S subunits; 50S subunits; ribosome structure; ribosome topography; mass spectrometry
Transfer ribonucleic acids (tRNAs) are challenging to identify and quantify from unseparated mixtures. Our lab previously developed the signature digestion approach for identifying tRNAs without specific separation. Here we describe the combination of relative quantification via enzyme-mediated isotope labeling with this signature digestion approach for the relative quantification of tRNAs. These quantitative signature digestion products were characterized using liquid chromatography mass spectrometry (LC-MS), and we find that up to 5-fold changes in tRNA abundance can be quantified from sub-microgram amounts of total tRNA. Quantitative tRNA signature digestion products must (i) incorporate an isotopic label during enzymatic digestion; (ii) have no m/z interferences from other signature digestion products in the sample and (iii) yield a linear response during LC-MS analysis. Under these experimental conditions, the RNase T1, A and U2 signature digestion products that potentially could be used for the relative quantification of Escherichia coli tRNAs were identified, and the linearity and sequence identify of RNase T1 signature digestion products were experimentally confirmed. These RNase T1 quantitative signature digestion products were then used in proof-of-principle experiments to quantify changes arising due to different culturing media to 17 tRNA families. This method enables new experiments where information regarding tRNA identity and changes in abundance are desired.
Methylthiotransferases (MTTases) are a closely related family of proteins that perform both radical-S-adenosylmethionine (SAM) mediated sulfur insertion and SAM-dependent methylation to modify nucleic acid or protein targets with a methyl thioether group (–SCH3). Members of two of the four known subgroups of MTTases have been characterized, typified by MiaB, which modifies N6-isopentenyladenosine (i6A) to 2-methylthio-N6-isopentenyladenosine (ms2i6A) in tRNA, and RimO, which modifies a specific aspartate residue in ribosomal protein S12. In this work, we have characterized the two MTTases encoded by Bacillus subtilis 168 and find that, consistent with bioinformatic predictions, ymcB is required for ms2i6A formation (MiaB activity), and yqeV is required for modification of N6-threonylcarbamoyladenosine (t6A) to 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A) in tRNA. The enzyme responsible for the latter activity belongs to a third MTTase subgroup, no member of which has previously been characterized. We performed domain-swapping experiments between YmcB and YqeV to narrow down the protein domain(s) responsible for distinguishing i6A from t6A and found that the C-terminal TRAM domain, putatively involved with RNA binding, is likely not involved with this discrimination. Finally, we performed a computational analysis to identify candidate residues outside the TRAM domain that may be involved with substrate recognition. These residues represent interesting targets for further analysis.
Pseudouridine, the so-called fifth nucleoside due to its ubiquitous presence in RNAs, remains among the most challenging modified nucleosides to characterize. As an isomer of the major nucleoside uridine, pseudouridine cannot be detected by standard reverse-transcriptase based DNA sequencing or RNase mapping approaches. Thus, over the past 15 years, investigators have focused on the unique structural properties of pseudouridine to develop selective derivatization or fragmentation strategies for its determination. While the N-cyclohexyl-N’-β-(4-methylmorpholinium) ethylcarbodiimide p-tosylate (CMCT)-reverse transcriptase assay remains both a popular and powerful approach to screen for pseudouridine in larger RNAs, mass spectrometry-based approaches are poised to play an increasingly important role in either confirming the findings of the CMCT-reverse transcriptase assay or in characterizing pseudouridine sequence placement and abundance in smaller RNAs. This review includes a brief discussion of pseudouridine including a summary of its biosynthesis and known importance within various RNAs. The review then focuses on chemical derivatization approaches that can be used to selectively modify pseudouridine to improve its detection, and the development of mass spectrometry-based assays for the identification and sequencing of pseudouridine in various RNAs.
CMCT derivatization; cyanoethylation; Pseudouridine synthase; MALDI-MS; LC-MS; modified nucleosides
Injection molded poly(methylmethacrylate) (IM-PMMA), chips were evaluated as potential candidates for capillary electrophoresis disposable chip applications. Mass production and usage of plastic microchips depends on chip-to-chip reproducibility and on analysis accuracy. Several important properties of IM-PMMA chips were considered: fabrication quality evaluated by environmental scanning electron microscope imaging, surface quality measurements, selected thermal/electrical properties as indicated by measurement of the current versus applied voltage (I–V) characteristic, and the influence of channel surface treatments. Electroosmotic flow was also evaluated for untreated and O2 reactive ion etching (RIE) treated surface microchips. The performance characteristics of single lane plastic microchip capillary electrophoresis (MCE) separations were evaluated using a mixture of two dyes - fluorescein (FL) and fluorescein isothiocyanate (FITC). To overcome non-wettability of the native IM-PMMA surface, a modifier, polyethylene oxide was added to the buffer as a dynamic coating. Chip performance reproducibility was studied for chips with and without surface modification via the process of RIE with O2 and by varying the hole position for the reservoir in the cover plate or on the pattern side of the chip. Additionally, the importance of reconditioning steps to achieve optimal performance reproducibility was also examined. It was found that more reproducible quantitative results were obtained when normalized values of migration time, peak area and peak height of FL and FITC were used instead of actual measured parameters
Injection molding; poly(methylmethacrylate) (PMMA); microchip electophoresis; reproducibility; characterization
Flow manipulation in sweeping microchip capillary electrophoresis (CE) is complicated by the free liquid communication between channels at the intersection, especially when the electroosmotic flows are mismatched in the main channel. Sweeping in traditional CE with cationic micelles is an effective way to concentrate anionic analytes. However, it is a challenge to transfer this method onto microchip CE because the dynamic coating process on capillary walls by cationic surfactants is interrupted when the sample solution free of surfactants is introduced into the microchip channels. This situation presents a difficulty in the sample loading, injection and dispensing processes. By adding surfactant at a concentration around the critical micelle concentration and by properly designing the voltage configuration, the flows in a microchip were effectively manipulated and this sweeping method was successfully moved to microchip CE using tetradecyltrimethylammonium bromide (TTAB). The sweeping effect of cationic surfactant in the sample solution was discussed theoretically and studied experimentally in traditional CE. The flows in a microchip were monitored with fluorescence imaging, and the injection and sweeping processes were studied by locating the detection point along the separation channel. A detection enhancement of up to 500-fold was achieved for 5-carboxyfluorescein.
Sweeping; Microchip; Capillary electrophoresis; Cationic surfactant; Flow control
A protocol that utilizes matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and N-cyclohexyl-N′-β-(4-methylmorpholinium)ethylcarbodiimide (CMC) derivatization to detect the post-transcriptionally modified nucleoside, pseudouridine, in RNA has been optimized for RNase digests. Because pseudouridine is mass-silent (i.e. the mass of pseudouridine is the same as the mass of uridine), after CMC derivatization and alkaline treatment, all pseudouridine residues exhibit a mass shift of 252 Da that allows its presence to be easily detected by mass spectrometry. This protocol is illustrated by the direct MALDI-MS identification of pseudouridines within Escherichia coli tRNATyrII starting from microgram amounts of sample. During this optimization study, it was discovered that the post-transcriptionally modified nucleoside 2-methylthio-N6-isopentenyladenosine, which is present in bacterial tRNAs, also retains a CMC unit after derivatization and incubation with base. Thus, care must be exercised when applying this MALDI-based CMC-derivatization approach for pseudouridine detection to samples containing transfer RNAs to minimize the misidentification of pseudouridine.
CMCT derivatization; Endonucleases; RNase T1; Pseudouridine; 2-methylthio-N6-isopentenyladenosine; tRNA; MALDI-MS; RNA signature products
A protocol has been developed that allows protein identifications using available DNA-based or protein sequences from a reference strain of a bacterial species to be extended to bacterial strains for which no prior DNA-based or protein sequence information exists. The protocol is predicated on careful isolation of a specific sub-cellular group of proteins. In this study, ribosomal proteins were chosen due to their high relative abundance and similarity in copy number per cell. After isolation of ribosomal proteins, MALDI-MS is used to acquire accurate protein molecular weights. An iterative comparison of reference protein molecular weights and identities is made to the resulting data, allowing for the straightforward identification of ribosomal proteins from any non-reference strains. This approach can reveal differences between proteins at the amino acid or post-translational level. The protocol was developed, validated and applied to ribosomal proteins from three strains of the extreme thermophile Thermus thermophilus. This approach revealed that nearly 60% of the ribosomal proteins from all three strains are identical. The extension of protein identification to additional bacterial strains can be useful in phylogenetic studies as well as in biomarker identification.
MALDI-TOF MS; Posttranslational modification; Ribosome; Subcellular proteomics; Thermus thermophilus; Top-down Proteomics
Patients suffering from cystic fibrosis (CF) commonly harbor the important pathogen Pseudomonas aeruginosa in their airways. During chronic late-stage CF, P. aeruginosa is known to grow under reduced oxygen tension and is even capable of respiring anaerobically within the thickened airway mucus, at a pH of ∼6.5. Therefore, proteins involved in anaerobic metabolism represent potentially important targets for therapeutic intervention. In this study, the clinically relevant “anaerobiome” or “proteogenome” of P. aeruginosa was assessed. First, two different proteomic approaches were used to identify proteins differentially expressed under anaerobic versus aerobic conditions. Microarray studies were also performed, and in general, the anaerobic transcriptome was in agreement with the proteomic results. However, we found that a major portion of the most upregulated genes in the presence of NO3− and NO2− are those encoding Pf1 bacteriophage. With anaerobic NO2−, the most downregulated genes are those involved postglycolytically and include many tricarboxylic acid cycle genes and those involved in the electron transport chain, especially those encoding the NADH dehydrogenase I complex. Finally, a signature-tagged mutagenesis library of P. aeruginosa was constructed to further screen genes required for both NO3− and NO2− respiration. In addition to genes anticipated to play important roles in the anaerobiome (anr, dnr, nar, nir, and nuo), the cysG and dksA genes were found to be required for both anaerobic NO3− and NO2− respiration. This study represents a major step in unraveling the molecular machinery involved in anaerobic NO3− and NO2− respiration and offers clues as to how we might disrupt such pathways in P. aeruginosa to limit the growth of this important CF pathogen when it is either limited or completely restricted in its oxygen supply.