Andrew C. Briscoe

Bio: Andrew C. Briscoe is an academic researcher from Boston Children's Hospital. The author has contributed to research in topics: Proteome & Glycomics. The author has an hindex of 8, co-authored 8 publications receiving 240 citations.

Optimizing sample handling for urinary proteomics.

Abstract: Interrogation of the urinary proteome for clinically useful biomarkers of disease will require normalization of methods for protein extraction and sample handling. Variations in collection methods and other procedures may introduce significant discrepancies in qualitative and quantitative measurements. Here we demonstrate that the method of protein extraction, length of handling at room temperature, and repetitive freeze−thaw cycles do not seem to alter the urinary proteome at either the protein or peptide level in a manner that degrades information obtainable by mass spectrometry.

One-Step Sample Concentration, Purification, and Albumin Depletion Method for Urinary Proteomics

Abstract: Workflows in urinary proteomics studies are often complex and require many steps to enrich, purify, deplete, and separate the complex mixture. Many of these methods are laborious, are time-consuming, and have the potential for error. Although individual steps of these methods have been previously studied, their downstream compatibilities with fractionation technologies such as off-gel electrophoresis have not been investigated. We developed a one-step sample preparation workflow that simultaneously (i) concentrates proteins, (ii) purifies by removing salts and other low molecular weight compounds, and (iii) depletes (albumin) from urine samples. This simple and robust workflow can be multiplexed and is compatible with a diverse range of downstream multidimensional separation technologies. Additionally, because of its high reproducibility and flexibility in processing samples with different volumes and concentrations, it has the potential to be used for standardization of urinary proteomics studies, as well as for studying other body fluids of similar complexity.

Identification of novel non-invasive biomarkers of urinary chronic pelvic pain syndrome: findings from the Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network.

Abstract: The idiopathic nature of Urological Chronic Pelvic Pain Syndrome (UCPPS), which encompasses interstitial cystitis/bladder pain syndrome (IC/BPS) and chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), has prompted an intense search for clinical biomarkers. Identification of clinically relevant, validated biologic markers of UCPPS has great potential to inform our understanding of UCPPS pathophysiology, development, and progression, and ultimately clinical management. To date, the cause of this syndrome is unknown and there is no ability to determine who is at risk of progression or who will respond to specific therapy. Accurate noninvasive tests, based on objective, specific, and definable levels of validated biomarkers, are essential to improve and standardized clinical guidelines and for more targeted management strategies based on patient profiles. The MAPP Research Network was established by the NIDDK of the NIH and represents a comprehensive approach to the study of UCPPS 1, 2. A major goal of this initiative is to better understand pathophysiology and identify potential therapeutic targets. In this MAPP Network study, we have utilized a biologically-driven, candidate biomarker discovery strategy, grounded in the basic biochemistry and physiology of this syndrome, to evaluate candidate non-invasive biomarkers of UCPPS. Reviews of the literature have revealed a number of candidate protein biomarker targets. Increased VEGF levels have been detected in bladder biopsy samples from patients with IC compared to controls, with levels correlating with pain severity 3, 4. Prior studies also suggest an important connection between neovascularization and IC, in that new blood vessels in biopsy samples of IC patients showed significantly lower levels of pericyte coverage of the nascent endothelium, evidence consistent with the known association between high levels of VEGF and immature vessel formation 3. Moreover, VEGF originally discovered as VPF (Vascular Permeability Factor), 5, 6 is the most potent endogenous vascular permeability factor and may mediate the dysregulated vascular permeability postulated to be important in UCPPS 7–9. Given these associations, we chose to include VEGF and its receptor VEGFR1, as biomarker candidates. Inflammation has been suggested to be an underlying pathophysiologic mechanism in UCPPS 10 and increased concentrations of pro-inflammatory cytokines (IL-1β, TNF-α, IL-8) have been reported in patients with UCPPS as compared with controls 11, 12. In this respect, MMPs are of particular interest. Originally thought to be associated exclusively with tissue remodeling and destruction, it is now widely appreciated that MMPs play a role in a variety of biologic processes including cytokine and growth factor release, tumor growth and progression, angiogenesis and a number of inflammatory conditions 13–17. During the inflammatory response, MMPs and their complexes are released from connective tissue cells in response to pro-inflammatory cytokines. Upregulation of MMP activity results in the recruitment of pro-inflammatory cells to the site of tissue injury. MMPs have also been implicated in the regulation of the immune response because of their ability to cleave inflammatory mediators and stimulate the clearance of inflammatory cells 18–21. Given that MMPs may play an important role in UCPPS as a function of the inflammatory phenotype, abnormal vasculature and the dysregulation of ECM (extracellular matrix) turnover present in this and other potentially related diseases we included these proteins in our study. NGAL, also known as Lipocalin-2 (Lcn2), is upregulated in numerous chronic and acute inflammatory conditions, including chronic kidney disease, ulcerative colitis and myocardial infarction 22, 23. There exists an intricate interplay between NGAL and other inflammatory cytokines as well, such that NGAL upregulation can be triggered by multiple inflammatory cytokines, including TNF-α, IL-1β and IL-17 22, 23. Moreover, NGAL itself is a modulator of the levels of other inflammatory cytokines, as well as of the behavior of inflammatory leukocytes 24–26. In our laboratory, we have demonstrated that NGAL is a significant stimulator of VEGF levels in breast tumor cells and can also independently induce the epithelial to mesenchymal transition in breast cancer, as well as being a noninvasive biomarker of this disease 27–29. NGAL can also form a complex with MMP-9, representing a distinct protein complex referred to as MMP-9/NGAL complex. We have previously shown that when NGAL complexes with MMP-9, it protects MMP-9 from degradation preserving its enzyme activity 16, 30. The MMP-9/NGAL complex has also been detected in urine samples from patients with various diseases. Based on these findings, we chose to measure NGAL as well as MMP-9/NGAL complex in the urine of individuals with UCPPS and correlate them with clinical symptoms. Here, we report sex-specific comparisons of candidate biomarkers among UCPPS and control cohorts to gain new insights into underlying pathophysiology. In addition, associations of biomarkers with symptom severity within the UCPPS cohort are examined. We anticipate that the identified biological markers may serve as potential candidates for further clinical evaluation, including in the development of new treatment strategies directed toward novel targets, as objective measures for patient classification schemes, and evaluation of clinical outcomes, as well as further characterization of underlying mechanism.

Addressing accuracy and precision issues in iTRAQ quantitation

Abstract: iTRAQ (isobaric tags for relative or absolute quantitation) is a mass spectrometry technology that allows quantitative comparison of protein abundance by measuring peak intensities of reporter ions released from iTRAQ-tagged peptides by fragmentation during MS/MS. However, current data analysis techniques for iTRAQ struggle to report reliable relative protein abundance estimates and suffer with problems of precision and accuracy. The precision of the data is affected by variance heterogeneity: low signal data have higher relative variability; however, low abundance peptides dominate data sets. Accuracy is compromised as ratios are compressed toward 1, leading to underestimation of the ratio. This study investigated both issues and proposed a methodology that combines the peptide measurements to give a robust protein estimate even when the data for the protein are sparse or at low intensity. Our data indicated that ratio compression arises from contamination during precursor ion selection, which occurs at a consistent proportion within an experiment and thus results in a linear relationship between expected and observed ratios. We proposed that a correction factor can be calculated from spiked proteins at known ratios. Then we demonstrated that variance heterogeneity is present in iTRAQ data sets irrespective of the analytical packages, LC-MS/MS instrumentation, and iTRAQ labeling kit (4-plex or 8-plex) used. We proposed using an additive-multiplicative error model for peak intensities in MS/MS quantitation and demonstrated that a variance-stabilizing normalization is able to address the error structure and stabilize the variance across the entire intensity range. The resulting uniform variance structure simplifies the downstream analysis. Heterogeneity of variance consistent with an additive-multiplicative model has been reported in other MS-based quantitation including fields outside of proteomics; consequently the variance-stabilizing normalization methodology has the potential to increase the capabilities of MS in quantitation across diverse areas of biology and chemistry.

Quantitative analysis of the intra- and inter-individual variability of the normal urinary proteome.

Abstract: Urine is a readily and noninvasively obtainable body fluid. Mass spectrometry (MS)-based proteomics has shown that urine contains thousands of proteins. Urine is a potential source of biomarkers for diseases of proximal and distal tissues but it is thought to be more variable than the more commonly used plasma. By LC-MS/MS analysis on an LTQ-Orbitrap without prefractionation we characterized the urinary proteome of seven normal human donors over three consecutive days. Label-free quantification of triplicate single runs covered the urinary proteome to a depth of more than 600 proteins. The median coefficient of variation (cv) of technical replicates was 0.18. Interday variability was markedly higher with a cv of 0.48 and the overall variation of the urinary proteome between individuals was 0.66. Thus technical variability in our data was 7.5%, whereas intrapersonal variability contributed 45.5% and interpersonal variability contributed 47.1% to total variability. Determination of the normal fluctuation of individual urinary proteins should be useful in establishing significance thresholds in biomarker studies. Our data also allowed definition of a common and abundant set of 500 proteins that were readily detectable in all studied individuals. This core urinary proteome has a high proportion of secreted, membrane, and relatively high-molecular weight proteins.

A comprehensive map of the human urinary proteome.

Abstract: The study of the human urinary proteome has the potential to offer significant insights into normal physiology as well as disease pathology. The information obtained from such studies could be applied to the diagnosis of various diseases. The high sensitivity, resolution, and mass accuracy of the latest generation of mass spectrometers provides an opportunity to accurately catalog the proteins present in human urine, including those present at low levels. To this end, we carried out a comprehensive analysis of human urinary proteome from healthy individuals using high-resolution Fourier transform mass spectrometry. Importantly, we used the Orbitrap for detecting ions in both MS (resolution 60 000) and MS/MS (resolution 15 000) modes. To increase the depth of our analysis, we characterized both unfractionated as well as lectin-enriched proteins in our experiments. In all, we identified 1823 proteins with less than 1% false discovery rate, of which 671 proteins have not previously been reported as constitue...

Physiological conditions can be reflected in human urine proteome and metabolome.

Abstract: Biomarkers are the measurable changes associated with physiological or pathophysiological processes. Urine, unlike blood, lacks mechanisms for maintaining homeostasis: it is therefore an ideal source of biomarkers that can reflect systemic changes. Urinary proteome and metabolome have been studied for their diagnostic capabilities, ability to monitor disease and prognostic utility. In this review, the effects of common physiological conditions such as gender, age, diet, daily rhythms, exercise, hormone status, lifestyle and extreme environments on human urine are discussed. These effects should be considered when biomarker studies of diseases are conducted. More importantly, if physiological changes can be reflected in urine, we have reason to expect that urine will become widely used to detect small and early changes in pathological and/or pharmacological conditions.