Norman H. L. Chiu

Rapid differentiation of in vitro cellular responses to toxic chemicals by using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Changes in protein expression as a cellular response to chemical exposure have been well established. Current methods for monitoring cellular responses usually require the use of specific reagents and/or labor-intensive procedures. The present study demonstrates the concept of using mass spectral pattern to distinguish different cellular responses. The concept is based on the ability to acquire a unique mass spectral pattern directly from a specific cell culture by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results demonstrate that distinguishable and reproducible spectral patterns can be obtained from different cellular responses.

Creating mass signatures for the detection of microRNA

MicroRNAs (miRNAs) play a crucial role in the post-transcriptional regulation of gene expression, and have already been associated with 160+ diseases. With the small sizes of miRNAs (19–25 nt) and the increasing number of human miRNAs (>2000) being reported, the accurate detection of a specific miRNA has continued to be a challenging issue. In this report, a new concept for improving the accuracy of direct mass spectrometric (MS) measurements of miRNAs is introduced. The concept aims at creating a unique signature in the mass spectrum of miRNA. To achieve this goal, one way is to capture a specific miRNA by using a complementary DNA probe. The miRNA is then eluted and digested with a specific endonuclease. The digested miRNA fragments are directly measured by using MS. The multiple peaks that correspond to the digested miRNA fragments are collectively defined as a mass signature of the miRNA being analyzed. Since the peak pattern is dependent on the intrinsic property, i.e. RNA sequence, of a specific miRNA, it is referred to as an intrinsic mass signature. Alternatively, a unique mass signature can be created by incorporating one or more extra nucleotides into the 3′ end of miRNA and the extended miRNA is measured by using MS. The molecular mass of the extended miRNA, which is defined as an extended mass signature, is expected to be different from the other miRNAs within the same sample. In comparison to matching the measured molecular mass of an undigested or unextended miRNA molecule to the expected molecular mass, the two different approaches to create a unique mass signature can improve the accuracy on qualitative MS detection of a specific miRNA.

Bottom-up mass spectrometric sequencing of microRNA

The increasing interest in microRNA (miRNA) as diagnostic biomarkers or potential drug targets has raised the demand for more accurate miRNA detection. One way to improve the accuracy is by using mass spectrometry (MS) to measure miRNA directly. Matrix-assisted laser desorption/ionization (MALDI) MS stands apart from other MS techniques due to the fact that a MALDI matrix is required for sample preparation. In this study, by exploiting the acidity of MALDI matrix and its mixing with miRNA prior to MS measurements, a simple method to generate RNA sequencing ladders is developed. The method utilizes the MALDI matrix to hydrolyze RNA at high temperature. The resulting sequencing ladders are ready to be measured without any desalting. By using MALDI SpiralTOF MS, the monoisotopic mass of each RNA fragment was measured. The RNA sequence was determined by sequentially comparing nucleotide compositions that were calculated from measured monoisotopic masses. The use of nucleotide compositions to assist the spectral interpretation has the advantages on distinguishing the complementary sequencing ladders, and allows the nucleotide identity at each position to be crosschecked multiple times. Together with the analysis of both complementary sequencing ladders, 100% sequence coverage and sequence accuracy were achieved in a blinded study.

Fluorometric cell-based assay for β-galactosidase activity in probiotic gram-positive bacterial cells - Lactobacillus helveticus.

Although methods for measuring β-galactosidase activity in intact gram-negative bacterial cells have been reported, the methods may not be applicable to measuring β-galactosidase activity in gram-positive bacterial cells. This report focuses on the development of a fluorometric cell-based assay for measuring β-galactosidase activity in gram-positive cells.

Carbon nanodots interference with lactate dehydrogenase assay in human monocyte THP-1 cells

BackgroundCarbon nanodots (CD), a new class of carbon nanomaterials with sizes below 10 nm, have recently attracted wide attention due to their superiority in water solubility, chemical inertness, and resistance to photobleaching. As a result, CD has found important and wide applications in energy, catalysis, biological labeling, bioimaging and drug delivery. On the other hand, due to the lack of available toxicity data, there is a growing concern regarding the potential risks of CD. Hence, accurate assessment of the cytotoxicity of CD has become more important than ever before. The lactate dehydrogenase (LDH) assay is widely used to detect cytotoxicity of various nanoparticles including CD. Many recent studies used LDH assay to study the CD toxicity in various cells. However, these studies failed to further examine whether the CD were interfering with the LDH assay which would alter their findings.FindingsThis study investigated the possible interference of carbon nanodots on the LDH assay in human monocyte THP-1 cells. Monocytes are known to be involved in inflammable vascular diseases, and have been suggested to be the targets for CD exposure. In this study, the cytotoxicity of CD in concentrations ranging from 0.075 to 0.60 mg/mL, was determined by using the LDH assay. To validate the results of LDH assay, the cell counting method with trypan blue staining was used. With 24 hours incubation time, the cell viability of THP-1 was significantly decreased according to the trypan blue staining method. Whereas, in the LDH assay, the CD was found to interfere in a dose-dependent manner with the NADH absorbance measurements at 340 nm.ConclusionsThis study represents the first report on the negative interference of CD on LDH assay, and caution should be observed when evaluating the cytotoxicity of CD.

Comparison of Accuracy on DNA Quantitation Determined by MALDI-TOF Mass Spectrometry and UV Spectrometry

ABSTRACT Although the UV absorbance of DNA at 260 nm has been recognized as a standard method for DNA quantitation, there are limitations of using UV spectrometry to determine the purity and identity of DNA. Recently, MALDI-TOF MS has proven to be an accurate technique for qualitative DNA analysis. In this study, the accuracy of MALDI-TOF MS for determining the concentration of DNA is evaluated and compared with that of the standard UV method. The results indicated that the accuracy of quantitative MALDI-TOF MS was comparable to that of the standard UV method and that measured DNA concentrations correlated well with those determined by the standard UV method.

High Percentage of Isomeric Human MicroRNA and Their Analytical Challenges

MicroRNA (miR) are short non-coding RNAs known to post-transcriptionally regulate gene expression, and have been reported as biomarkers for various diseases. miR have also been served as potential drug targets. The identity, functions and detection of a specific miR are determined by its RNA sequence, whose composition is made up of only 4 canonical ribonucleotides. Hence, among over two thousand human miR, their nucleotide compositions are expected to be similar but the extent of similarity has not been reported. In this study, the sequences of mature human miR were downloaded from miRBase, and collated using different tools to determine and compare their nucleotide compositions and sequences. 55% of all human miR were found to be structural isomers. The structural isomers of miR (SimiR) are defined as having the same size and identical nucleotide composition. A number of SimiR were also found to have high sequence similarities. To investigate the extent of SimiR in biological samples, three disease models were chosen, and disease-associated miR were identified from miR2Disease. Among the disease models, as high as 73% of miR were found to be SimiR. This report provides the missing information about human miR and highlights the challenges on the detection of SimiR.

Isolation and Detection of Carcinogenic Nucleic Acid Adducts

In general, nucleic acid adducts are formed when harmful chemical compounds react covalently with cellular DNA or RNA molecules. With only four natural nucleobases in DNA or RNA, identical nucleic acid adducts can theoretically occur at multiple positions within the human genome or transcriptome. The frequency of nucleic acid adduction is further increased by the reactivity of DNA or RNA to form adducts with many different types of chemicals, which include both exogenous compounds that our bodies have been exposed and endogenous compounds that are generated through normal metabolic activities in our bodies.1,2 Some exogenous compounds may require metabolic activation prior to the formation of nucleic acid adducts, whereas others may react directly with nucleic acids. If DNA adducts are not effectively removed by the DNA repair mechanism, the adducts can directly interfere with DNA replication and transcription.3 Similarly, the presence of adducts in RNA molecules can affect their biological functions. From the results of many experimental studies, the association of either DNA adducts or RNA adducts to cancer have already been well established. In the case of DNA adducts, it is widely recognized as the key element for the onset of carcinogenesis. Both DNA adducts and RNA adducts are, therefore, important biomarkers for cancer research, which include the monitoring of exposure to carcinogens, genetic mutation, DNA repair and so on. Similar to the analysis of other cancer biomarkers, both identification and quantification of nucleic acid adducts are required. Prior to the detection of nucleic acid adducts, it is important to ensure the biological or clinical samples are collected and stored properly. Equally important, the isolation of genomic DNA or RNA has to be carried out with high efficiency, which includes the yield and purity of selected material, reproducibility and the rate of sample throughput. For the detection of nucleic acid adducts, the requirements can be divided into the following order:

Nanospray desorption electrospray ionization mass spectrometry of untreated and treated probiotic Lactobacillus reuteri cells

AbstractMass spectrometry has proven to be a useful technique for rapid identification of bacterial cells. Among various ionization techniques in mass spectrometry, matrix-assisted laser desorption/ionization (MALDI) has been commonly used for the identification of bacterial cells. Recently, MALDI mass spectrometry has also been utilized to distinguish cellular responses. Ambient ionization techniques do support whole bacterial cell analysis, which include desorption electrospray ionization (DESI). Nanospray DESI (nDESI) is a new variant of DESI, and its application to whole-cell mass spectrometry is limited. In this project, the use of nDESI mass spectrometry to measure probiotic Lactobacillus reuteri (LR) cells is explored. A unique and reproducible mass spectral pattern of untreated LR cells was obtained by using 50% methanol/water as nDESI solvent. The use of nDESI mass spectrometry is further extended to distinguish untreated LR cells from treated LR cells that have been exposed to low pH. These findings demonstrate the feasibility of using nDESI in whole-cell mass spectrometry. Graphical abstractᅟ

MicroRNA MultiTool: A Software for Identifying Modified and Unmodified Human microRNA Using Mass Spectrometry

microRNA (miRNA) are short endogenous non-coding RNA that play a crucial role in post-transcriptional gene regulation and have been implicated in the initiation and progression of 160+ human diseases. Excellent analytical methods have been developed for the measurement of miRNA by mass spectrometry. However, interpretation of mass spectrometric data has been an incapacitating bottleneck in miRNA identification. This study details the development of MicroRNA MultiTool, a software for the identification of miRNA from mass spectrometric data. The software includes capabilities such as miRNA search and mass calculator, modified miRNA mass calculator, and miRNA fragment search. MicroRNA MultiTool bridges the gap between experimental data and identification of miRNA by providing a rapid means of mass spectrometric data interpretation.