Identification and characterization of the Thermus thermophilus 5-methylcytidine (m5C) methyltransferase modifying 23 S ribosomal RNA (rRNA) base C1942.

Methylation of cytidines at carbon-5 is a common posttranscriptional RNA modification encountered across all domains of life. Here, we characterize the modifications of C1942 and C1962 in Thermus thermophilus 23 S rRNA as 5-methylcytidines (m(5)C) and identify the two associated methyltransferases. The methyltransferase modifying C1942, named RlmO, has not been characterized previously. RlmO modifies naked 23 S rRNA, but not the assembled 50 S subunit or 70 S ribosomes. The x-ray crystal structure of this enzyme in complex with the S-adenosyl-l-methionine cofactor at 1.7 Å resolution confirms that RlmO is structurally related to other m(5)C rRNA methyltransferases. Key residues in the active site are located similar to the further distant 5-methyluridine methyltransferase RlmD, suggestive of a similar enzymatic mechanism. RlmO homologues are primarily found in mesophilic bacteria related to T. thermophilus. In accordance, we find that growth of the T. thermophilus strain with an inactivated C1942 methyltransferase gene is not compromised at non-optimal temperatures.

Mass spectrometry in the biology of RNA and its modifications.

Many powerful analytical techniques for investigation of nucleic acids exist in the average modern molecular biology lab. The current review will focus on questions in RNA biology that have been answered by the use of mass spectrometry, which means that new biological information is the purpose and outcome of most of the studies we refer to. The review begins with a brief account of the subject "MS in the biology of RNA" and an overview of the prevalent RNA modifications identified to date. Fundamental considerations about mass spectrometric analysis of RNA are presented with the aim of detailing the analytical possibilities and challenges relating to the unique chemical nature of nucleic acids. The main biological topics covered are RNA modifications and the enzymes that perform the modifications. Modifications of RNA are essential in biology, and it is a field where mass spectrometry clearly adds knowledge of biological importance compared to traditional methods used in nucleic acid research. The biological applications are divided into analyses exclusively performed at the building block (mainly nucleoside) level and investigations involving mass spectrometry at the oligonucleotide level. We conclude the review discussing aspects of RNA identification and quantifications, which are upcoming fields for MS in RNA research. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.

3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine is one of two novel post-transcriptional modifications in tRNALys(UUU) from Trypanosoma brucei.

tRNA is the most heavily modified of all RNA types, with typically 10-20% of the residues being post-transcriptionally altered. Unravelling the modification pattern of a tRNA is a challenging task; there are 92 currently known tRNA modifications, many of which are chemically similar. Furthermore, the tRNA has to be investigated with single-nucleotide resolution in order to ensure complete mapping of all modifications. In the present work, we characterized tRNA(Lys)(UUU) from Trypanosoma brucei, and provide a complete overview of its post-transcriptional modifications. The first step was MALDI-TOF MS of two independent digests of the tRNA, with RNase A and RNase T1, respectively. This revealed digestion products harbouring mass-changing modifications. Next, the modifications were mapped at the nucleotide level in the RNase products by tandem MS. Comparison with the sequence of the unmodified tRNA revealed the modified residues. The modifications were further characterized at the nucleoside level by chromatographic retention time and fragmentation pattern upon higher-order tandem MS. Phylogenetic comparison with modifications in tRNA(Lys) from other organisms was used through the entire analysis. We identified modifications on 12 nucleosides in tRNA(Lys)(UUU), where U47 exhibited a novel modification, 3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine, based on identical chromatographic retention and MS fragmentation as the synthetic nucleoside. A37 was observed in two versions: a minor fraction with the previously described 2-methylthio-N(6)-threonylcarbamoyl-modification, and a major fraction with A37 being modified by a 294.0-Da moiety. The latter product is the largest adenosine modification reported so far, and we discuss its nature and origin.

Glycopeptide profiling of beta-2-glycoprotein I by mass spectrometry reveals attenuated sialylation in patients with antiphospholipid syndrome.

Beta2-glycoprotein I (beta2GPI) is a five-domain protein associated with the antiphospholipid syndrome (APS), however, its normal biological function is yet to be defined. beta2GPI is N-glycosylated at several asparagine residues and the glycan moiety conjugated to residue 143 has been proposed to interact with the Gly40-Arg43 motif of beta2GPI. The Gly40-Arg43 motif has also been proposed to serve as the epitope for the anti-beta2GPI autoantibody associated with APS. We hypothesized that the structure or composition of the glycan at Asn-143 might be associated with the APS symptom by shielding or exposing the Gly40-Arg43 motif towards the anti-beta2GPI autoantibody. To test this hypothesis we used mass spectrometry (MS) for comparative glycopeptide profiling of human beta2GPI obtained from blood serum from four healthy test subjects and six APS patients. It revealed significant differences in the extent of sialylation and branching of glycans at Asn-143. Biantennary glycans were more abundant than triantennary glycans at Asn-143 in both healthy subjects and patients. In APS patient samples we observed a decrease in sialylated triantennary glycans and an increase in sialylated biantennary glycan structures, as compared to controls. These data indicate that some APS patients have beta2GPI molecules with a reduced number of negatively charged sialic acid units in the glycan structure at Asn-143. This alteration of the electrostatic properties of the glycan moiety may attenuate the intramolecular interactions with the positively charged Gly40-Arg43 motif of beta2GPI and, in turn, leads to conformational instability and exposure of the disease-related linear epitope Gly40-Arg43 to the circulating autoantibody. Thus, our study suggests a link between site-specific glycan profiles of beta2GPI and the pathology of antiphospholipid syndrome.

1-Hydroxypyrene as a biomarker of PAH exposure in the marine polychaete Nereis diversicolor.

The possibility of using the pyrene metabolite 1-hydroxypyrene as a biomarker of polycyclic aromatic hydrocarbons (PAHs) exposure was investigated by exposure of the marine polychaete Nereis diversicolor to several PAHs in the laboratory. Animals were exposed to pyrene alone and to five different PAHs - phenanthrene, anthracene, pyrene, benzo[a]pyrene and benzo[k]flouranthene. After five days of exposure the concentrations of parent PAHs and 1-hydroxypyrene were identified using three different analytical methods, high-performance liquid chromatography with fluorescence detection (HPLC/F), synchronous fluorescence spectroscopy (SFS) and gas chromatography with mass spectrometric detection (GC/MS). The SFS measurements of 1-hydroxypyrene were validated by the more sensitive method HPLC/F. The positive correlation between total PAHs and 1-hydroxypyrene concentrations in the polychaete tissues observed in experiments, suggests the feasibility of 1-hydroxypyrene as a suitable biomarker for total PAH exposure assessment. Furthermore, the possibility of employment of the simple and rapid SFS method instead of HPLC/F for biomarker analysis has been confirmed by the positive and significant correlation between results achieved by these two analytical methods.

Towards liquid chromatography time-scale peptide sequencing and characterization of post-translational modifications in the negative-ion mode using electron detachment dissociation tandem mass spectrometry.

Electron detachment dissociation (EDD) of peptide poly-anions is gentle towards post-translational modifications (PTMs) and produces predictable and interpretable fragment ion types (a., x ions). However, EDD is considered an inefficient fragmentation technique and has not yet been implemented in large-scale peptide characterization strategies. We successfully increased the EDD fragmentation efficiency (up to 9%), and demonstrate for the first time the utility of EDD-MS/MS in liquid chromatography time-scale experiments. Peptides and phosphopeptides were analyzed in both positive- and negative-ion mode using electron capture/transfer dissociation (ECD/ETD) and EDD in comparison. Using approximately 1 pmol of a BSA tryptic digest, LC-EDD-MS/MS sequenced 14 peptides (27% aa sequence coverage) and LC-ECD-MS/MS sequenced 19 peptides (39% aa sequence coverage). Seven peptides (18% aa sequence coverage) were sequenced by both EDD and ECD. The relative small overlap of identified BSA peptides demonstrates the complementarity of the two dissociation modes. Phosphopeptide mixtures from three trypsin-digested phosphoproteins were subjected to LC-EDD-MS/MS resulting in the identification of five phospho-peptides. Of those, one was not found in a previous study using a similar sample and LC-ETD-MS/MS in the positive-ion mode. In this study, the ECD fragmentation efficiency (15.7% av.) was superior to the EDD fragmentation efficiency (3.6% av.). However, given the increase in amino acid sequence coverage and extended PTM characterization the new regime of EDD in combination with other ion-electron fragmentation techniques in the positive-ion mode is a step towards a more comprehensive strategy of analysis in proteome research.

Investigating the biomarker potential of glycoproteins using comparative glycoprofiling - application to tissue inhibitor of metalloproteinases-1.

Cancer-induced alterations of protein glycosylations are well-known phenomena. Hence, the glycoprofile of certain glycoproteins can potentially be used as biomarkers for early diagnosis. However, there are a substantial number of candidates and the techniques for measuring their biomarker potential are limited, calling for new methods. Here, we have investigated the cancer marker potential of the glycoprofile of tissue inhibitor of metalloproteinase-1 (TIMP-1) using a method for comparative glycoprofiling. Glycoprofiles were obtained from plasma TIMP-1 of five healthy donors and five colorectal cancer (CRC) patients showing increased amounts of TIMP-1. Furthermore, the TIMP-1 glycoprofiles of media from two colon cancer cell lines (CCC) and a prostate cancer cell line were determined as disease references. TIMP-1 was purified from IgG-depleted samples using immuno affinity and gel electrophoresis and the glycoprofiling was performed using glycopeptide enrichment and mass spectrometry. The heterogeneous glycoprofiles of TIMP-1 were found to be highly conserved among the healthy donors, proving an ideal candidate marker and showed high reproducibility of the method. Numerous CCC-specific TIMP-1 glycans were observed illustrating cancer-induced changes. Unexpectedly, quantitation revealed that the glycoprofiles of healthy donors and CRC patients varied minimally. Considering the increased CRC TIMP-1 levels and the observed CCC-specific glycans, the lack of variation indicates that the increased amount of CRC TIMP-1 is not a direct product of the cancer cells. Hence, the TIMP-1 glycoprofile holds no biomarker potential for CRC when using plasma as the sample origin. This study clearly illustrates that the technique is capable of performing individualised site-specific glycan analysis and representing a new tool for biomarker investigation of low-abundant glycoproteins.

Biotransformation of polycyclic aromatic hydrocarbons in marine polychaetes.

Deposit-feeding polychaetes constitute the dominant macrofauna in marine environments that tend to be depositional centers for organic matter and contaminants. Polychaetes are known to accumulate polycyclic aromatic hydrocarbons (PAHs) from both particulate and dissolved phases but less is known about the mechanisms underlying elimination of accumulated PAHs. An important pathway of elimination is through biotransformation which results in increased aqueous solubility of the otherwise hydrophobic PAHs. Biotransformation in marine polychaetes proceeds in a two phased process similar to those well studied in vertebrates, phase I enzymes belonging to the Cytochrome P450 (CYP) enzyme family, along with a few phase II enzymes have been identified in marine polychaetes. In this review we aim at highlighting advances in the mechanistic understanding of PAH biotransformation in marine polychaetes by including data obtained using analytical chemistry and molecular techniques. In marine polychaetes induction of CYP enzyme activity after exposure to PAHs and the mechanism behind this is currently not well established. Conflicting results regarding the inducibility of CYP enzymes from polychaetes have led to the suggestion that induction in polychaetes is mediated through a different mechanistic pathway, which is corroborated by the apparent lack of an AhR homologous in marine polychaetes. Also, none of the currently identified CYP genes from marine polychaetes are isoforms of those regulated by the AhR in vertebrates. Relatively few studies of phase II enzymes in marine polychaetes are currently available and most of these studies have not measured the activity of specific phase II enzymes and identified phase II metabolites but used an extraction technique only allowing determination of the overall amount of phase II metabolites. Studies in insects and various marine invertebrates suggest that in invertebrates, enzymes in the important phase II enzyme family, UDP-glucuronosyl transferases primarily use glucoside as co-substrate as opposed to the vertebrate cosubstrate glucuronic acid. Recent studies in marine polychaetes have however identified glucuronidation of PAHs indicating no mechanistic difference in co-substrate preference among UDP-glucuronosyl transferases between vertebrates and marine polychaetes but it might suggest a mechanistic difference between marine polychaetes and insects.

Peptide sequencing and characterization of post-translational modifications by enhanced ion-charging and liquid chromatography electron-transfer dissociation tandem mass spectrometry.

We have tested the effect of m-nitrobenzyl alcohol (m-NBA) as a method to increase the average charge state of protonated gas-phase molecular ions generated by ESI from tryptic peptides and phosphopeptides. Various concentrations of m-NBA were added to the mobile phases of a liquid chromatography system coupled to an ESI tandem mass spectrometer. Addition of just 0.1% m-NBA changed the average charge state for the identified tryptic BSA peptides from 2.2+ to 2.6+. As a result, the predominant charge states for BSA peptides were changed from 2+ to > or =3+. To evaluate the benefits of peptide charge enhancement, the ETD fragmentation efficiency and Mascot peptide score were compared for BSA peptides in charge states 2+ and 3+. In all cases but one, triply charged peptides fragmented more efficiently than the analogues 2+ peptide ions. On average, triply charged peptides received a 68% higher Mascot score (24 units) than doubly charged peptides. m-NBA also increased the average charge state of phosphopeptides by up to 0.5 charge unit. The ease of implementation and the analytical benefits of charge enhancement of tryptic peptides by addition of m-NBA to the LC solvents suggest the general application of this reagent in proteomic studies that employ ETD-MS/MS and related techniques.

Limited microbial degradation of pyrene metabolites from the estuarine polychaete Nereis diversicolor.

We compared microbial mineralization of [4,5,9,10-14C]pyrene and its eukaryotic [4,5,9,10-14C]pyrene metabolites in estuarine sediments. Metabolites were obtained by exposing the estuarine deposit-feeding polychaete Nereis diversicolor to sediment-associated 14C-pyrene, followed by homogenization of the worms and extraction of the pyrene-metabolites. In sediment from a pristine Danish Fjord only 2.6% of the added metabolite-label and 1.7% of the pyrene-label were mineralized to 14CO2 during 175 days incubation. Pre-exposure of the pristine sediment to unlabelled pyrene for 60 days increased the mineralization potential for 14C-pyrene substantially, as 81.2% was mineralized to 14CO2 during 95 days incubation, whereas 14C-pyrene metabolite label was unaffected by pre-exposure to pyrene. In comparison, naturally aged bunker-oil contaminated sediment did not show elevated potentials for mineralization of neither 14C-pyrene nor 14C-metabolites. Six bacterial strains of known pyrene degraders were tested for growth on crystalline 1-hydroxypyrene. 1-Hydroxypyrene is the only intermediate eucaryotic metabolite of pyrene. The results indicate that 1-hydroxypyrene was not utilized as a sole source of carbon and energy by any of them. In addition, respiration was depressed in all six strains when exposed to crystalline 1-Hydroxypyrene, demonstrating an acute toxic effect of 1-hydroxypyrene. The results presented here suggest that microbial degradation of pyrene is not enhanced by release of aqueous and polar metabolites by marine invertebrates.

Synchronous fluorescence spectrometry of 1-hydroxypyrene: a rapid screening method for identification of PAH exposure in tissue from marine polychaetes.

The uptake of polycyclic aromatic hydrocarbons (PAHs) by marine deposit-feeding invertebrates can be determined by screening for PAH-derived metabolites. We identified 1-hydroxypyrene as the only intermediate metabolite in tissue of four species of deposit-feeding polychaetes, Nereis diversicolor, Nereis virens, Arenicola marina, and Capitella sp. I exposed to pyrene spiked sediment. Synchronous fluorescence spectroscopy (SFS) provides a fast and simple method for both qualitative and quantitative analysis of 1-hydroxypyrene in all four species. The SFS assay was validated using HPLC with ultraviolet detection. A good correlation between 1-hydroxypyrene concentrations determined by the two methods was observed. We used HPLC with fluorescence detection combined with enzymatic hydrolysis of conjugated metabolites to investigate species specific metabolite patterns. A tentative aqueous metabolite identification scheme indicates that Nereid polychaetes predominately make use of glucuronide conjugation whereas Capitella sp. I. and Arenicola marina appear to utilize predominantly sulfate and/or glucoside conjugation. The usefulness of 1-hydroxypyrene as a biomarker for PAH exposure in deposit-feeding invertebrates is discussed.

1-Hydroxypyrene glucuronide as the major aqueous pyrene metabolite in tissue and gut fluid from the marine deposit-feeding polychaete Nereis diversicolor.

Both 1-hydroxypyrene and 1-hydroxypyrene glucuronide are identified as the primary phase I and phase II metabolites of the four-ringed polycyclic aromatic hydrocarbon (PAH) pyrene in the marine deposit-feeding polychaete Nereis diversicolor. Identification of pyrene and primary metabolites was performed using high-pressure liquid chromatography (HPLC) with diode-array detection and fluorescence detection (HPLC/DAD/F) and an ion-trap mass spectrometer for positive identification of 1-hydroxypyrene glucuronide. Besides 1-hydroxypyrene and 1-hydroxypyrene glucuronide, the HPLC/F trace of tissue samples from pyrene-exposed worms showed three additional low-intensity peaks that may be related to pyrene metabolism based on similar excitation/emission wavelengths. The peaks were all too low in intensity to be positively identified. Of the total PAH in tissue, 1-hydroxypyrene glucuronide, 1-hydroxypyrene, and pyrene constituted 73%, 2%, and 25% respectively. Gut elimination of metabolic products is supported by the identification of 1-hydroxypyrene and 1-hydroxypyrene glucuronide in both gut fluid and defecation water. Being the only phase I metabolite of pyrene, 1-hydroxypyrene becomes a useful marker for PAH exposure, and it may serve as a valuable model compound for assessing species-specific PAH metabolic capabilities.

Identification of 1-hydroxypyrene glucuronide in tissue of marine polychaete Nereis diversicolor by liquid chromatography/ion trap multiple mass spectrometry.

1-Hydroxypyrene glucuronide is identified as the single major aqueous metabolite of the tetracyclic aromatic hydrocarbon pyrene, in tissue from a deposit-feeding marine polychaete, Nereis diversicolor. Identification was performed using an ion trap mass spectrometer fitted with an atmospheric pressure chemical ionization (APCI) probe and connected to a high-performance liquid chromatography/diode array detector (HPLC/DAD) system. Besides 1-hydroxypyrene, the 339-nm UV trace of tissue samples from pyrene-exposed worms showed only one dominant peak that could be related to pyrene metabolism. Negative APCI-MS of this supposed 1- hydroxypyrene conjugate gave a characteristic signal at m/z 429 corresponding to the molecular ion of 1-hydroxypyrene glucuronide plus eluent adducts ([M - H + 2H(2)O](-)). Fragmentation pathways were studied by isolating the abundant ion at m/z 429 in the ion trap and performing multiple mass spectrometric experiments (MS(n)). The fragmentations observed were consistent with the proposed identification. Two low intensity LC peaks that could be related to pyrene metabolism by their DAD absorption spectra were also present in the 339-nm UV chromatogram of tissue samples. However, these peaks could not be identified by their mass spectra in negative ion mode due to ion suppression by very abundant co-eluting impurities. The present method shows that LC/MS(n) is a fast and useful analytical tool for identification of aqueous polycyclic aromatic hydrocarbon biotransformation products in samples from relatively small marine invertebrates with limited sample preparation.