Daniel B. Martin

Circulating microRNAs as stable blood-based markers for cancer detection

Improved approaches for the detection of common epithelial malignancies are urgently needed to reduce the worldwide morbidity and mortality caused by cancer. MicroRNAs (miRNAs) are small (≈22 nt) regulatory RNAs that are frequently dysregulated in cancer and have shown promise as tissue-based markers for cancer classification and prognostication. We show here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. miRNAs originating from human prostate cancer xenografts enter the circulation, are readily measured in plasma, and can robustly distinguish xenografted mice from controls. This concept extends to cancer in humans, where serum levels of miR-141 (a miRNA expressed in prostate cancer) can distinguish patients with prostate cancer from healthy controls. Our results establish the measurement of tumor-derived miRNAs in serum or plasma as an important approach for the blood-based detection of human cancer.

Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry.

Quantitative proteome profiling using stable isotope protein tagging and automated tandem mass spectrometry (MS/MS) is an emerging technology with great potential for the functional analysis of biological systems and for the detection of clinical diagnostic or prognostic marker proteins. Owing to the enormous complexity of proteomes, their comprehensive analysis is an as-yet-unresolved technical challenge. However, biologically or clinically important information can be obtained if specific, information-rich protein classes, or sub-proteomes, are isolated and analyzed. Glycosylation is the most common post-translational modification. Here we describe a method for the selective isolation, identification and quantification of peptides that contain N-linked carbohydrates. It is based on the conjugation of glycoproteins to a solid support using hydrazide chemistry, stable isotope labeling of glycopeptides and the specific release of formerly N-linked glycosylated peptides via peptide- N-glycosidase F (PNGase F). The recovered peptides are then identified and quantified by MS/MS. We applied the approach to the analysis of plasma membrane proteins and proteins contained in human blood serum.

A guided tour of the Trans‐Proteomic Pipeline

The Trans‐Proteomic Pipeline (TPP) is a suite of software tools for the analysis of MS/MS data sets. The tools encompass most of the steps in a proteomic data analysis workflow in a single, integrated software system. Specifically, the TPP supports all steps from spectrometer output file conversion to protein‐level statistical validation, including quantification by stable isotope ratios. We describe here the full workflow of the TPP and the tools therein, along with an example on a sample data set, demonstrating that the setup and use of the tools are straightforward and well supported and do not require specialized informatic resources or knowledge.

Complexity of the microRNA repertoire revealed by next-generation sequencing.

MicroRNAs (miRNAs) have been implicated to play key roles in normal physiological functions, and altered expression of specific miRNAs has been associated with a number of diseases. It is of great interest to understand their roles and a prerequisite for such study is the ability to comprehensively and accurately assess the levels of the entire repertoire of miRNAs in a given sample. It has been shown that some miRNAs frequently have sequence variations termed isomirs. To better understand the extent of miRNA sequence heterogeneity and its potential implications for miRNA function and measurement, we conducted a comprehensive survey of miRNA sequence variations from human and mouse samples using next generation sequencing platforms. Our results suggest that the process of generating this isomir spectrum might not be random and that heterogeneity at the ends of miRNA affects the consistency and accuracy of miRNA level measurement. In addition, we have constructed a database from our sequencing data that catalogs the entire repertoire of miRNA sequences (http://galas.systemsbiology.net/cgi-bin/isomir/find.pl). This enables users to determine the most abundant sequence and the degree of heterogeneity for each individual miRNA species. This information will be useful both to better understand the functions of isomirs and to improve probe or primer design for miRNA detection and measurement.

Sustained activation of XBP1 splicing leads to endothelial apoptosis and atherosclerosis development in response to disturbed flow

X-box binding protein 1 (XBP1) is a key signal transducer in endoplasmic reticulum stress response, and its potential role in the atherosclerosis development is unknown. This study aims to explore the impact of XBP1 on maintaining endothelial integrity related to atherosclerosis and to delineate the underlying mechanism. We found that XBP1 was highly expressed at branch points and areas of atherosclerotic lesions in the arteries of ApoE−/− mice, which was related to the severity of lesion development. In vitro study using human umbilical vein endothelial cells (HUVECs) indicated that disturbed flow increased the activation of XBP1 expression and splicing. Overexpression of spliced XBP1 induced apoptosis of HUVECs and endothelial loss from blood vessels during ex vivo cultures because of caspase activation and down-regulation of VE-cadherin resulting from transcriptional suppression and matrix metalloproteinase-mediated degradation. Reconstitution of VE-cadherin by Ad-VEcad significantly increased Ad-XBP1s-infected HUVEC survival. Importantly, Ad-XBP1s gene transfer to the vessel wall of ApoE−/− mice resulted in development of atherosclerotic lesions after aorta isografting. These results indicate that XBP1 plays an important role in maintaining endothelial integrity and atherosclerosis development, which provides a potential therapeutic target to intervene in atherosclerosis.

Quantitative proteomic analysis of proteins released by neoplastic prostate epithelium.

Prostate cancer is unusual among neoplasms in that it may be diagnosed at a curable stage through detection of a protein in serum, the serine protease prostate-specific antigen (PSA). PSA is secreted by both normal and neoplastic prostate epithelial cells in response to androgenic hormones and has found widespread use in cancer screening. Because PSA screening is controversial due to sensitivity and specificity issues, efforts continue to focus on the identification and characterization of additional markers that may be used for diagnostic and therapeutic purposes. In this study, we report the application of quantitative proteomic techniques that incorporate isotope coded affinity tag reagents and tandem mass spectrometry to comprehensively identify secreted and cell surface proteins from neoplastic prostate epithelium. LNCaP cells, a prostate tumor-derived cell line that secretes PSA in response to androgen exposure, were grown in a low protein-defined media under androgen-stimulated (A+) and -starved (A-) conditions. Proteomic analysis of the media identified in excess of 600 proteins, 524 of which could be quantified. Nine percent of the proteins had A+/A- ratios > 2.0, including PSA, and 2.5% had ratios < 0.5. A subset of these androgen-regulated proteins appeared to be expressed in abundance. Of these, selected mass spectrometry observations were confirmed by Western analysis. The findings suggest that androgen-mediated release of proteins may occur through the activation of proteolytic enzymes rather than exclusively through transcriptional or translational control mechanisms. On the basis of their known functional roles, several of the abundant androgen-regulated proteins may participate in the progression of neoplastic epithelial cell growth and should be considered as potential serum markers of neoplastic prostate diseases.

Prevalence of transcription promoters within archaeal operons and coding sequences.

Despite the knowledge of complex prokaryotic‐transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well‐defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome‐wide characterization of transcript structures of ∼64% of all genes, including putative non‐coding RNAs in Halobacterium salinarum NRC‐1. Our integrative analysis of transcriptome dynamics and protein–DNA interaction data sets showed widespread environment‐dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3′ ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes—events usually considered spurious or non‐functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements.

Advances in quantitative proteomics using stable isotope tags

A great deal of current biological and clinical research is directed at the interpretation of the information contained in the human genome sequence in terms of the structure, function and control of biological systems and processes. Proteomics, the systematic analysis of proteins, is becoming a critical component in this endeavor because proteomic measurements are carried out directly on proteins--the catalysts and effectors of essentially all biological functions. To detect changes in protein profiles that might provide important diagnostic or functional insights, proteomic analyses necessarily have to be quantitative. This article summarizes recent technological advances in quantitative proteomics.

DirecTag: Accurate Sequence Tags from Peptide MS/MS through Statistical Scoring

In shotgun proteomics, tandem mass spectra of peptides are typically identified through database search algorithms such as Sequest. We have developed DirecTag, an open-source algorithm to infer partial sequence tags directly from observed fragment ions. This algorithm is unique in its implementation of three separate scoring systems to evaluate each tag on the basis of peak intensity, m/ z fidelity, and complementarity. In data sets from several types of mass spectrometers, DirecTag reproducibly exceeded the accuracy and speed of InsPecT and GutenTag, two previously published algorithms for this purpose. The source code and binaries for DirecTag are available from http://fenchurch.mc.vanderbilt.edu.

MRMer, an Interactive Open Source and Cross-platform System for Data Extraction and Visualization of Multiple Reaction Monitoring Experiments

Multiple reaction monitoring (MRM) mass spectrometry identifies and quantifies specific peptides in a complex mixture with very high sensitivity and speed and thus has promise for the high throughput screening of clinical samples for candidate biomarkers. We have developed an interactive software platform, called MRMer, for managing highly complex MRM-MS experiments, including quantitative analyses using heavy/light isotopic peptide pairs. MRMer parses and extracts information from MS files encoded in the platform-independent mzXML data format. It extracts and infers precursor-product ion transition pairings, computes integrated ion intensities, and permits rapid visual curation for analyses exceeding 1000 precursor-product pairs. Results can be easily output for quantitative comparison of consecutive runs. Additionally MRMer incorporates features that permit the quantitative analysis experiments including heavy and light isotopic peptide pairs. MRMer is open source and provided under the Apache 2.0 license.