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Biomedical subjects

Patrick A Limbach

Publications and source records attributed to Patrick A Limbach.

At least 19 recordsLinked to original sources
PubMed2026

Dietary effects on cytosolic and mitochondrial tRNA abundance and modification patterns across mouse tissues.

Transfer RNAs (tRNAs) are central to protein synthesis and are increasingly recognized as dynamic regulators of gene expression whose abundance and chemical modifications are subject to precise biological control. Here, we systematically investigate how two distinct dietary interventions, low-protein and high-fat diets, reshape the tRNA landscape across multiple mouse tissues, using RNA mass spectrometry and ordered two-template relay sequencing (OTTR-seq) to comprehensively profile cytosolic and mitochondrial tRNAs at single-nucleotide resolution. We reveal pronounced tissue-specific biases in tRNA isodecoder expression, including the unexpected presence of full-length cytosolic tRNAs in mature sperm with a distinct isotype composition. In somatic tissues such as liver and heart, dietary conditions alter both tRNA abundance and key modifications known to regulate decoding efficiency, whereas in reproductive tissues diet primarily affects the abundance of select tRNAs with comparatively limited changes in modification profiles. We further demonstrate that mitochondrial tRNAs are subject to diet-responsive changes in both abundance and modification status and that even subtle differences in dietary fat composition are sufficient to alter tRNA modification signatures. Together, these findings establish the tRNA epitranscriptome as a sensitive and tissue-specific sensor of nutritional state and provide a resource for understanding how dietary cues interface with translational regulation in somatic and reproductive tissues.

Male↗
PubMed2025

A survey of dietary effects on tRNA abundance and modifications.

Transfer RNAs (tRNAs) play a central role in protein translation and are increasingly recognized as dynamic regulators of gene expression. Both physiological and environmental signals can modulate tRNA abundance and chemical modifications, yet the impact of dietary cues on the tRNA landscape remains poorly understood. Here, we investigated the effects of two distinct dietary interventions-low-protein and high-fat diets-on tRNA abundance and modification profiles across multiple mouse tissues. We conducted a comprehensive analysis of tRNA abundance and modification changes in response to these nutritional challenges using RNA mass spectrometry and Ordered Two-Template Relay sequencing (OTTR-seq), a modified-base-sensitive tRNA sequencing method. Our results reveal both shared and tissue-specific alterations in abundance and modifications of specific nuclear and mitochondrial genome-encoded tRNAs in response to dietary conditions at isotype, isoacceptor, and isodecoder levels. As many of the tissue-specific or diet-responsive tRNA modifications have been previously reported to affect decoding efficiency or translational fidelity, these results have implications for understanding translational adaptation in response to dietary conditions.

Journal Article↗
PubMed2006

Mass spectrometry-based detection of transfer RNAs by their signature endonuclease digestion products.

The separation of biologically active, pure, and specific tRNAs is difficult due to the overall similarity in secondary and tertiary structures of different tRNAs. Because prior methods do not facilitate high-resolution separations of the extremely complex mixture represented by a cellular tRNA population, global studies of tRNA identity and/or abundance are difficult. We have discovered that the enzymatic digestion of an individual tRNA by a ribonuclease (e.g., RNase T1) will generate digestion products unique to that particular tRNA, and we show that a comparison of an organism's complete complement of tRNA RNase digestion products yields a set of unique or "signature" digestion product(s) that ultimately enable the detection of individual tRNAs from a total tRNA pool. Detection is facilitated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and proof-of-principle is demonstrated on the whole tRNA pool from Escherichia coli. This method will enable the individual identification of tRNA isoacceptors without requiring specific affinity purification or extensive chromatographic and/or electrophoretic purification. Further, experimental identifications of tRNAs or other RNAs will now be possible using this signature digestion product approach in a manner similar to peptide mass fingerprinting used in proteomics, allowing RNomic studies of RNA at the post-transcriptional level.

Escherichia coli↗
PubMed2006

Unlimited-volume electrokinetic stacking injection in sweeping capillary electrophoresis using a cationic surfactant.

Sweeping is an effective and convenient way for online sample preconcentration in micellar electrokinetic chromatography. The usual procedure includes a hydrodynamic injection step carried out by applying pressure to the sample vial followed by the subsequent sweeping and separation processes. The injected sample volume is limited by the dimensions of the capillary because a part of the capillary has to be left free of sample solution for the subsequent sweeping and separation steps. In addition, when a short capillary, such as 4-10 cm, is used for sweeping, the injected sample volume is small even if the entire capillary is filled with sample solution. To solve this problem, an electrokinetic stacking injection (EKSI) scheme was developed by using a cationic surfactant, dodecyltrimethylammonium bromide, for sweeping in capillary electrophoresis. An experimental model was proposed, and the entire process was theoretically analyzed. According to the theoretical discussion, the optimal conditions for two model analytes, 5-carboxyfluorescein (5-FAM) and sodium fluorescein (FL), were experimentally determined. The injected sample plug lengths for 5-FAM and FL under 20.1 kV for 60 min were experimentally estimated as 836 and 729 cm, corresponding to 28- and 24-fold the effective capillary length, respectively. The EKSI scheme resulted in increased detection factors for 5-FAM and FL of 4.5 x 10(3) and 4.0 x 10(3) using 60-min injection relative to a traditional pressure injection.

Cations↗
PubMed2006

On-line sample preconcentration by sweeping with dodecyltrimethylammonium bromide in capillary zone electrophoresis.

On-line sample preconcentration of oligonucleotides with a new sweeping carrier was developed by using dodecyltrimethylammonium bromide (DTAB) below the critical micelle concentration (CMC). The sweeping results with DTAB below and above the CMC were compared. The use of DTAB below the CMC benefits the preconcentration of the oligonucleotides, while the use of DTAB above the CMC is good for hydrophobic small molecules. The factors affecting the sweeping results were optimized and this method was evaluated by constructing calibration curves for thrombin aptamers. The sweeping scheme produced a 112-fold sensitivity enhancement for the oligonucleotides relative to that run in a running buffer without DTAB. The sweeping method developed here can be a good reinforcement of the preconcentration scheme by sweeping when less-hydrophobic analytes or large negatively-charged molecules need to be preconcentrated.

Acetonitriles↗
PubMed2006

On-line sample preconcentration using field-amplified stacking injection in microchip capillary electrophoresis.

Previous reports describing sample stacking on microchip capillary electrophoresis (microCE) have regarded the microchip channels as a closed system and treated the bulk flow as in traditional capillary electrophoresis. This work demonstrates that the flows arising from the intersection should be investigated as an open system. It is shown that the pressure-driven flows into or from the branch channels due to bulk velocity mismatch in the main channel should not be neglected but can be used for liquid transportation in the channels. On the basis of these concepts, a sample preconcentration scheme was developed in a commercially available single-cross glass chip for microCE. Similar to field-amplified stacking injection in traditional CE, a low conductivity sample buffer plug was introduced into the separation channel immediately before the negatively charged analyte molecules were injected. The detection sensitivity was improved by 94-, 108-, and 160-fold for fluorescein-5-isothiocyanate, fluorescein disodium, and 5-carboxyfluorescein, respectively, relative to a traditional pinched injection. The calibration curves for fluorescein and 5-carboxyfluorescein demonstrated good linearity in the concentration range (1-60 nM) investigated with acceptable reproducibility of migration time and peak height and area ratios (4-5% RSD). This preconcentration scheme will be of particular significance to the practical use of microCE in the emerging miniaturized analytical instrumentation.

Buffers↗
PubMed2006

Mass spectrometry of RNA: linking the genome to the proteome.

Ribonucleic acids (RNAs) are continuing to attract increased attention as they are found to play pivotal roles in biological systems. Just as genomics and proteomics have been enabled by the development of effective analytical techniques and instrumentation, the large-scale analysis of non-protein coding (nc)RNAs will benefit as new analytical methodologies, such as mass spectrometry (MS), are developed for their analysis. Mass spectrometry offers a number of advantages for RNA analysis arising from its ability to provide mass and sequence information starting with limited amounts of sample. This review will highlight recent developments in the field of MS that enable the characterization of RNA modification status, RNA tertiary structures, and ncRNA expression levels. These developments will also be placed in perspective of how MS of RNAs can help elucidate the link between the genome and proteome.

Animals↗
PubMed2006

Analytical performance of polymer-based microfluidic devices fabricated by computer numerical controlled machining.

A study comparing the electrophoretic separation performance attainable from microchips molded by masters fabricated using conventional CNC machining techniques with commercial microchips, wire imprinted microchips, and microchips from LIGA molding devices is presented. An electrophoresis-based detection system using fluorescence microscopy was used to determine the analytical utility of these microchips. The separation performance of CNC microchips was comparable to commercially available microchips as well as those fabricated from LIGA masters. The important feature of the CNC machined masters is that they have rapid design-to-device times using routinely available machining tools. This low-cost prototyping approach provides a new entry point for researchers interested in thermoplastic microchips and can accelerate the development of polymer-based lab-on-a-chip devices.

Computer-Aided Design↗
PubMed2006

Characterizing the reproducibility of a protein profiling method for the analysis of mouse bronchoalveolar lavage fluid.

The detection of biomarkers in biological fluids has been advanced by the introduction of mass spectrometry screening methods such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), which enables the detection of the presence and the molecular mass of proteins in unfractionated mixtures. The generation of reproducible mass spectra over the course of an experiment is vital in obtaining data in which differences in protein profiles between diseased and healthy states can be assessed correctly. We have developed a protocol to automate the collection of protein profiling data from a large number of samples using MALDI-TOFMS, and we used these samples to characterize the technical reproducibility of the method. This protocol has been used for the analysis of proteins found in bronchoalveolar lavage fluid samples from mice with the ultimate goal of enabling the discovery of differential expression patterns predictive of the development of chronic obstructive pulmonary disease. Samples were purified using magnetic bead-based technology and analyzed on an AnchorChip target plate. Our results demonstrate that the number of peaks detected reproducibly decreases significantly as sample size increases, which motivates the need for technical replicates to be explicitly included in the analysis of MALDI-TOF-based protein profiling studies.

Acrolein↗
PubMed2006

Bisphenol A facilitates bypass of androgen ablation therapy in prostate cancer.

Prostatic adenocarcinomas depend on androgen for growth and survival. First line treatment of disseminated disease exploits this dependence by specifically targeting androgen receptor function. Clinical evidence has shown that androgen receptor is reactivated in recurrent tumors despite the continuance of androgen deprivation therapy. Several factors have been shown to restore androgen receptor activity under these conditions, including somatic mutation of the androgen receptor ligand-binding domain. We have shown previously that select tumor-derived mutants of the androgen receptor are receptive to activation by bisphenol A (BPA), an endocrine-disrupting compound that is leached from polycarbonate plastics and epoxy resins into the human food supply. Moreover, we have shown that BPA can promote cell cycle progression in cultured prostate cancer cells under conditions of androgen deprivation. Here, we challenged the effect of BPA on the therapeutic response in a xenograft model system of prostate cancer containing the endogenous BPA-responsive AR-T877A mutant protein. We show that after androgen deprivation, BPA enhanced both cellular proliferation rates and tumor growth. These effects were mediated, at least in part, through androgen receptor activity, as prostate-specific antigen levels rose with accelerated kinetics in BPA-exposed animals. Thus, at levels relevant to human exposure, BPA can modulate tumor cell growth and advance biochemical recurrence in tumors expressing the AR-T877A mutation.

Adenocarcinoma↗
PubMed2005

Association of the 17-kDa extrinsic protein with photosystem II in higher plants.

The structural association of the spinach 17-kDa extrinsic protein of photosystem II with other extrinsic and membrane-bound components of the photosystem was investigated by labeling the 17-kDa extrinsic protein with the amino-group-specific reagent N-hydroxysuccinimidobiotin both on intact photosystem II membranes or as a free protein in solution. After isolation of the biotinylated molecules, the modified 17-kDa proteins were allowed to rebind to photosystem II membranes which were depleted of the 17-kDa component. Differential binding of the protein biotinylated in solution compared to unmodified 17-kDa protein or 17-kDa protein modified on PS II membranes was observed. This indicated possible steric or ionic interference because of biotinylated lysyl residues present on the protein modified in solution. Biotinylated sites on the different modified 17-kDa proteins were identified by trypsin and Staphylococcus V8 protease digestion, followed by affinity chromatography enrichment of the biotinylated peptides and analysis of the peptide fragment mixture by nanospray liquid chromatography-tandem mass spectrometry. Four lysyl residues that were modified when the protein was biotinylated in solution were not biotinylated when the protein was modified on the PS II membrane (90K, 96K, 101K, and 102K). These residues appear to identify a protein domain involved in the interaction of the 17-kDa protein with the other components of the photosystem.

Amino Acid Sequence↗
PubMed2005

The autofluorescence of plastic materials and chips measured under laser irradiation.

Plastic materials have the potential to substitute for glass substrates used in microfluidic and microTAS systems adding flexibility in materials' choices. Optical quality plastic materials with a low autofluorescence are crucial for optimal detection by fluorescence and laser induced fluorescence techniques. This paper summarizes a series of optical investigations on commercially available plastic chip materials (PMMA, COC, PC, PDMS) and chips made from those materials. Intrinsic optical constants of plastic materials-refractive index for bulk materials-determined by spectroscopic ellipsometry and transmission spectroscopy in the visible range are presented. The laser-induced autofluorescence of materials and chips was assessed at four laser wavelengths, namely, 403, 488, 532 and 633 nm. Considerable bleaching of the autofluorescence was observed under continuous laser illumination. Overall, the longer wavelength laser excitation sources yielded less autofluorescence. PDMS exhibited the least autofluorescence and was comparable to BoroFloat glass. In all cases, chips exhibited slightly higher autofluorescence than the raw plastic materials from which they had been made.

Equipment Failure Analysis↗
PubMed2005

Estimation of pK(a) values using microchip capillary electrophoresis and indirect fluorescence detection.

Microchip capillary electrophoresis (CE), coupled with indirect fluorescence detection was investigated for estimating the pK(a) values of non-fluorescent compounds. The CE method is based on the differences in electrophoretic mobility of the analyte as a function of the pH of the running buffer. Nine compounds were tested, including several of pharmaceutical importance, with pK(a) values from 10.3 to 4.6. All buffers contained 5-TAMRA as the fluorescent probe for indirect detection. Calculated pK(a) values agreed well with literature values obtained by traditional methods, differing not more than 0.2 from the literature value. The current work on single lane chips demonstrates the principle of microchip CE with indirect detection as a viable method for estimating pK(a) values. However, increased throughput will be required using a multilane chip to enable the approach to be used practically.

Aniline Compounds↗
PubMed2005

Quantitation of ribonucleic acids using 18O labeling and mass spectrometry.

A previous limitation in the analysis of ribonucleic acids (RNAs) by mass spectrometry (MS) has been the inability to obtain quantitative information relating to total RNA, RNA subunits, and undermodified nucleosides in a straightforward manner. Here, a simple and rapid method has been developed for the relative quantitation of small RNAs using 18O labeling and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). One RNA sample is digested with RNase T1 in 18O-labeled ("heavy") water with the 18O being incorporated at the 3'-phosphate end of oligonucleotides upon hydrolysis. A second RNA sample is digested with RNase T1 in normal ("light") water. The two samples are then combined and analyzed by MALDI-MS. Relative ion abundances of the light- and heavy-water digestion products, which are separated by 2 Da due to the isotopic mass of 18O, reveal relative quantitation information from the two RNA samples. The accuracy and reproducibility of this approach were tested on 18 known RNA samples and 4 unknown RNA samples. The coefficients of variation for quantitation were found to be generally below 15% when using MALDI-MS. The approach yields accurate quantitative information for heavy-to-light ratios greater than 1:2. This method should prove useful for quantitatively characterizing variations in RNA production and variations in the amount of posttranscriptionally modified nucleosides.

Isotope Labeling↗
PubMed2005

Estimation of logP(ow) values for neutral and basic compounds by microchip microemulsion electrokinetic chromatography with indirect fluorimetric detection (muMEEKC-IFD).

Microchip microemulsion electrokinetic chromatography with indirect fluorimetric detection (muMEEKC-IFD) was used to obtain logP octanol/water (logP(ow)) values for neutral and basic compounds. Six compounds, with logP(ow) values between 0.38 and 5.03, were used to create a calibration curve relating the log of retention factors (logk) obtained from muMEEKC-IFD with the known logP(ow) values. The logP(ow) values for six additional compounds were determined using the logk values obtained by muMEEKC-IFD and the linear relationship between logP(ow) and logk established for the standard compounds. The muMEEKC-IFD buffer was composed of 50 mM 3-[cyclohexylamino]-1-propane-sulfonic acid (CAPS) buffer (pH 10.4) containing 1.2% n-heptane (v/v), 2% sodium dodecylsulfate (w/v), 8% 1-butanol (v/v) and 4 microM 5-carboxytetramethyl-rhodamine (TAMRA) as the fluorophore probe for indirect detection. The muMEEKC-IFD provided an accurate method for estimating logP(ow) values and also a means for analyzing compounds that are non-fluorescent.

Calibration↗
PubMed2005

Extending ribosomal protein identifications to unsequenced bacterial strains using matrix-assisted laser desorption/ionization mass spectrometry.

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.

Bacterial Proteins↗
PubMed2005

Interactions between sodium dodecyl sulfate micelles and peptides during matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of proteolytic digests.

Although sodium dodecyl sulfate (SDS) is routinely used as a denaturing agent for proteins, its presence is highly detrimental on the analysis of peptides and proteins by mass spectrometry. It has been found, however, that when SDS is present in concentrations near to or above its critical micelle concentration (CMC), improvements in the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of peptide mixtures or hydrophobic proteins are obtained. To elucidate possible explanations for such improvements, here we have undertaken a study examining the effect of SDS micelles on peptide mixtures. Fluorescently labeled peptides were used as probes to determine whether hydrophobic or hydrophilic peptides interact exclusively with SDS micelles. In addition, four globular proteins were digested with trypsin and then various amounts of SDS were added before MALDI mass spectrometry. To examine the role of mixture complexity on the mass spectral results, the tryptic digest of bovine serum albumin was also fractionated according to hydrophobicity before SDS treatment. Results from these experiments suggest that micelle-peptide interactions increase peptide-matrix cocrystallization irrespective of analyte hydrophobicity. As these studies were performed using the dried-droplet method of sample spotting, the presence of micelles is also hypothesized to reduce Marangoni effects during the crystallization process.

Micelles↗