Intact MicroRNA Analysis Using High Resolution Mass Spectrometry
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
2014; 25 (1): 80-87
Performance evaluation of affinity ligands for depletion of abundant plasma proteins.
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
2013; 939: 10-16
MicroRNAs (miRNAs) are small single-stranded non-coding RNAs that post-transcriptionally regulate gene expression, and play key roles in the regulation of a variety of cellular processes and in disease. New tools to analyze miRNAs will add understanding of the physiological origins and biological functions of this class of molecules. In this study, we investigate the utility of high resolution mass spectrometry for the analysis of miRNAs through proof-of-concept experiments. We demonstrate the ability of mass spectrometry to resolve and separate miRNAs and corresponding 3' variants in mixtures. The mass accuracy of the monoisotopic deprotonated peaks from various miRNAs is in the low ppm range. We compare fragmentation of miRNA by collision-induced dissociation (CID) and by higher-energy collisional dissociation (HCD) which yields similar sequence coverage from both methods but additional fragmentation by HCD versus CID. We measure the linear dynamic range, limit of detection, and limit of quantitation of miRNA loaded onto a C18 column. Lastly, we explore the use of data-dependent acquisition of MS/MS spectra of miRNA during online LC-MS and demonstrate that multiple charge states can be fragmented, yielding nearly full sequence coverage of miRNA on a chromatographic time scale. We conclude that high resolution mass spectrometry allows the separation and measurement of miRNAs in mixtures and a standard LC-MS setup can be adapted for online analysis of these molecules.
View details for DOI 10.1007/s13361-013-0759-x
View details for Web of Science ID 000329239600010
View details for PubMedID 24174127
Effects of cathepsin K deficiency on intercellular junction proteins, luminal mucus layers, and extracellular matrix constituents in the mouse colon.
2012; 393 (12): 1391-1403
Human plasma is a commonly used diagnostic fluid in clinical chemistry. In-depth plasma proteomic analysis is performed to search for disease biomarkers, however the large dynamic range of protein abundance in plasma presents a substantial analytical challenge. Removal of abundant plasma proteins using antibody capture approaches is a common and attractive means to reduce sample complexity and to aid the analysis of lower abundance proteins of interest. A novel class of heavy chain camelid-derived affinity ligands produced in Saccharomyces cerevisiae, has recently been developed as an alternative to antibody-based depletion methods. Here, we evaluate the performance characteristics of these ligands for removal of high abundance plasma proteins. Affinity ligands were tested for the removal of 14 abundant human plasma proteins. The performance characteristics were evaluated by gel-electrophoresis and LC-MS/MS of the bound and flow-through fractions. The capacity of a 5.6mL column was found to be 125μL of plasma. Replicate analysis demonstrated high column reproducibility and linearity, efficient removal of abundant proteins, and enrichment of lower abundance proteins observed after depletion. The novel class of affinity ligands provides an attractive alternative to traditional antibody-based immunodepletion methods.
View details for DOI 10.1016/j.jchromb.2013.09.008
View details for PubMedID 24090752
Studies of intestinal morphology and cathepsin B expression in a transgenic mouse aiming at intestine-specific expression of Cath B-EGFP
2011; 392 (11): 983-993
Cathepsin K has been shown to exhibit antimicrobial and anti-inflammatory activities in the mouse colon. To further elucidate its role, we used Ctsk-/- mice and demonstrated that the absence of cathepsin K was accompanied by elevated protein levels of related cysteine cathepsins (cathepsins B, L, and X) in the colon. In principle, such changes could result in altered subcellular localization; however, the trafficking of cysteine cathepsins was not affected in the colon of Ctsk-/- mice. However, cathepsin K deficiency affected the extracellular matrix constituents, as higher amounts of collagen IV and laminin were observed. Moreover, the localization pattern of the intercellular junction proteins E-cadherin and occludin was altered in the colon of Ctsk-/- mice, suggesting potential impairment of the barrier function. Thus, we used an ex vivo method for assessing the mucus layers and showed that the absence of cathepsin K had no influence on mucus organization and growth. The data of this study support the notion that cathepsin K contributes to intestinal homeostasis and tissue architecture, but the lack of cathepsin K activity is not expected to affect the mucus-depending barrier functions of the mouse colon. These results are important with regard to oral administration of cathepsin K inhibitors that are currently under investigation in clinical trials.
View details for DOI 10.1515/hsz-2012-0204
View details for PubMedID 23152408
Imaging of protease functions--current guide to spotting cysteine cathepsins in classical and novel scenes of action in mammalian epithelial cells and tissues.
Italian journal of anatomy and embryology = Archivio italiano di anatomia ed embriologia
2011; 116 (1): 1-19
Cathepsin B has been shown to not only reside within endo-lysosomes of intestinal epithelial cells, but it was also secreted into the extracellular space of intestinal mucosa in physiological and pathological conditions. In an effort to further investigate the function of this protease in the intestine, we generated a transgenic mouse model that would enable us to visualize the localization of cathepsin B in vivo. Previously we showed that the A33-antigen promoter could be successfully used in vitro in order to express cathepsin B-green fluorescent protein chimeras in cells that co-expressed the intestine-specific transcription factor Cdx1. In this study an analog approach was used to express chimeric cathepsin B specifically in the intestine of transgenic animals. No overt phenotype was observed for the transgenic mice that reproduced normally. Biochemical and morphological studies confirmed that the overall intestinal phenotype including the structure and polarity of this tissue as well as cell numbers and differentiation states were not altered in the A33-CathB-EGFP mice when compared to wild type animals. However, transgenic expression of chimeric cathepsin B could not be visualized because it was not translated in situ although the transgene was maintained over several generations.
View details for DOI 10.1515/BC.2011.096
View details for Web of Science ID 000295921900006
View details for PubMedID 21871011
Identification and analysis of mammalian KLK6 orthologue genes for prediction of physiological substrates
COMPUTATIONAL BIOLOGY AND CHEMISTRY
2008; 32 (2): 111-121
The human genome encodes some hundreds of proteases. Many of these are well studied and understood with respect to their biochemistry, molecular mechanisms of proteolytic cleavage, expression patterns, molecular structure, substrate preferences and regulatory mechanisms, including their endogenous inhibitors. Moreover, precise determination of protease localisation within subcellular compartments, peri- and extracellular spaces has been extremely useful in elucidating biological functions of peptidases. This can be achieved by refined methodology as will be demonstrated herein for the cysteine cathepsins. Besides localisation, it is now feasible to study in situ enzymatic activity at the various levels of subcellular compartments, cells, tissues, and even whole organisms including mouse.
View details for PubMedID 21898969
Human kallikrein-related peptidase 6 (KLK6) is a novel serine protease that is aberrantly expressed in human cancers and represents a serum biomarker for the molecular diagnosis and monitoring of ovarian cancer. Here, we report the cloning and analysis of human kallikrein-related peptidase 6 gene (KLK6) orthologues in model organisms and farm animals. The corresponding full-length cDNAs were assembled from partial sequences retrieved from EST and genomic databases. Alignment of inferred protein sequences indicated a high degree of conservation of the encoded enzyme. We found that, similarly to (HUMAN)KLK6, monkey, cattle, mouse and rat orthologue genes encode for multiple transcript variants. This strengthens our previously published data showing that (HUMAN)KLK6 transcription is coordinately regulated by alternative promoters. Analysis of the KLK6 upstream genomic region led to the identification of multiple conserved regulatory regions with motifs for nuclear receptor transcription factors. Interestingly, we found that specific CpG dinucleotides in the proximal promoter, that were shown to regulate (HUMAN)KLK6 gene expression via DNA methylation, are conserved in orthologue genes, indicating epigenetic regulation of the KLK6 gene. Construction of a protein-protein interaction network indicated that KLK6 likely acts on the TGF-b1 signal transduction pathway to regulate certain cytoskeletal proteins, such as vimentin and keratin 8, thus, KLK6 may control cell shape that, in turn, regulates cell migration and motility.
View details for DOI 10.1016/j.compbiolchem.2007.11.002
View details for Web of Science ID 000255263300004
View details for PubMedID 18243805