Speaker: Allen Everett, Johns Hopkins University
Topic: Proteomics Discovery of Circulating Pulmonary Hypertension Biomarkers: IGF binding proteins are associated with disease severity
Date: Monday, February 15th, 2021
Time: 1:00 PM Presentation
Location: Webinar – see emails on Feb. 4 and 11 for invite link. Join the mailing list
Background: Pulmonary arterial hypertension is a progressive and fatal disease characterized by sustained elevations of pulmonary artery pressure. We lack circulating, diagnostic and prognostic markers to improve outcomes and develop new therapies.
Methods and Results: We performed proteomics discovery using high resolution mass spectrometry to identify new circulating biomarkers of pulmonary arterial hypertension. Plasma samples from patients with idiopathic pulmonary arterial hypertension (N=9, age 35.2 ± 11.2 years, 89% female) and normal controls (N=9, age 34.8 ± 10.6 years, 100% female) were processed by liquid chromatography/tandem mass spectrometry. A total of 826 (0.047 False Discovery Rate) idiopathic pulmonary arterial hypertension and 461 (0.087 False Discovery Rate) control proteins were identified. By Volcano plot, 153 proteins showed > 2 fold change, P<0.05. Carbonic anhydrase 2 (CA2) and Insulin like growth factor binding protein (IGFBP2) were top molecules by spectral counts. When all IGF axis molecules were examined, spectral counts for IGF1, IGF2, IGFBP1, IGFBP4, and IGFBP7 were also different between PAH and control. ELISA verification (N=41 PAH and N=39 controls) demonstrated that IGF1 and 2 were decreased and IGFBP1, 2, 4, 5, 7 and CA2 were increased in PAH. In association with disease severity, IGFBP2, 4 and 7 were associated with decreased 6MWD and IGFBP1, 2, 5 associated with PVR. IGFBP2, 4, and 7 were associated with survival (Kaplan Meier). CA2 was not associated with clinical severity.
Conclusions: We identified candidate plasma proteins that can distinguish PAH from control and verified CA2 and multiple members of the IGF axis associated with PAH and PAH severity. Suggesting that the IGF axis may play an important role in PAH pathogenesis and may be an important diagnostic for PAH, response to therapy and play a role in the pathogenesis of PAH.
Speaker: Shao-En Ong, University of Washington
Topic: Kinome analyses for pharmacoproteomics
Date: Tuesday, January 19th, 2021
Time: 2:00 PM Presentation
Location: Webinar – see emails on Jan. 8 and 15 for invite link. Join the mailing list
Abstract: With few targeted therapies for genetic alterations in cancer, pharmacogenomics has been used to link genetic features with drug response. Because proteomics allows sensitive and direct measurements of cellular signaling pathways, we developed a novel pharmacoproteomics platform to identify kinase pathways correlating with drug response by combining kinobead-based activity profiling of 346 kinases and high-throughput screening of 299 kinase inhibitors in 17 hepatocellular carcinoma (HCC) cell lines. We identified novel kinases involved in drug resistance, that upon small molecule inhibition or genetic knockdown, rewired cellular signaling and restored chemosensitivity. We applied kinobead-MS in clinical HCC samples to identify signatures of drug sensitivity common to cell lines and patient tumors. Our broadly applicable approach identifies kinome features responsible for the activity of individual drugs and provides a resource for biomarker discovery and target deconvolution.
Speaker: Perry Wang, US Food and Drug Administration
Topic: Advanced Application of LC-MS and Challenges
Date: Monday, December 14th, 2020
Time: 2:00 pm Presentation
Location: Webinar – see emails on Dec. 3 and 10 for invite link. Join the mailing list
Abstract: Liquid chromatography-mass spectrometry (LC-MS) is the most sensitive analytical technique by far. It combines the physical separation power of liquid chromatography with the mass analysis capabilities of mass spectrometry. Because the individual capabilities of each technique are synergistically enhanced, the combination of liquid chromatography with mass spectrometry could be called a “perfect marriage” -liquid chromatography separates components in mixtures by affinity and mass spectrometry differentiates the components by mass. Therefore, LC-MS is applied in a broad field including biotechnology, environment monitoring, food safety, and pharmaceutical, agrochemical, and cosmetic industries. However, the technique often faces a great challenge -matrix effect, which can be observed as either a loss (ion suppression), or an increase (ion enhancement) in responses. The matrix effects affect the detection capability, precision and/or accuracy for the analytes of interest. Thus, the matrix effects should be evaluated during method development by comparing the response of a standard solution prepared in a sample matrix over the response in neat solutions or comparing the calibration-curve slope of standard solutions prepared in sample matrix over the slope of standards prepared in neat solutions. Unfortunately, a representative matrix is not always available for some studies, and how to evaluate and minimize the matrix effects are challenging. Different techniques to minimize matrix effects will be presented and the concept of matrix effect factor (MEF) will be introduced and discussed.
Speaker: Yansheng Liu, Yale University School of Medicine
Topic: DIA-MS and Its Application to Profiling Cellular Proteome and Proteoform Dynamics
Date: Monday, November 16th, 2020
Time: 2:00 pm Presentation
Location: Webinar – see emails on Nov. 5 and 12 for invite link. Join the mailing list
Abstract: The term ‘proteoform’ is now used to designate different molecular forms in which the protein product of a single gene can be found, including changes due to genetic variations, alternatively spliced RNA transcripts, and post-translational modifications. Although proteoform is normally studied by the top-down approach, we will discuss a new bottom-up strategy to investigate the site-specific modiforms. We will first review the data-independent acquisition mass spectrometry (DIA-MS) and its development in our group. We will then present how we use DIA-MS, pulse stable isotope-labeled amino acids in cells (pSILAC) approach, and genome-wide correlation analysis for quantifying both abundance and turnover rate of proteins in cancer cell models.
Speaker: Carlos Larriba-Andaluz, Indiana University–Purdue University Indianapolis
Topic: Understanding Ion Mobility Separation in High-Resolution Instruments. Caveats of and deviations from the Mason-Schamp Equation for small molecules
Date: Monday, October 19th, 2020
Time: 1:00 pm Presentation
Location: Webinar – see emails on Oct. 8 and 15 for invite link. Join the mailing list
Abstract: Ion mobility Spectrometry (IMS) is an analytical tool that has recently carried a great deal of interest in the field of Analytical Chemistry. As such, IMS is now ubiquitously present as an integrated part of many MS systems. This has resulted in major improvements in experimental setups, with very recent impressive achievements in terms of separation and peak capacity, alongside instruments like the Structure for Lossless Ion Manipulation (SLIM) or Field Asymmetric Waveform IMS (FAIMS) that have shown the capability of separating even isotopomers. Its progress is so remarkable that the theoretical ground commonly used to describe IMS has become insufficient to explain some experimentally observed separations; with some interpretations of this high-resolution separations remaining merely speculative at this point. It is therefore necessary to carefully analyze our common theoretical tools and describe the simplifications employed to arrive at equations such as the Mason-Schamp approximation so that a clear picture is provided on when these estimations may be employed. An effort is made to provide a concise and simple explanation of the simplifications that result on the Mason-Schamp equation. This is done from a method of moments perspective (up to the two-temperature theory) with an estimate of the error for different types of approximations. Based on this knowledge, a numerical tool, IMoS 2, that models an ion in a physical gas with the ability to stochastically calculate the drift velocity for a rotating ion under an arbitrary field, is proposed that may explain some of these separations recently observed.
Three main issues have been addressed in this work: 1) how the Collision Cross Section (CCS) is calculated and when its validity may be compromised due to rotation, moment of inertia or dipole alignment and other influences, 2) the effect of the electric field; when is it safe to ignore and when it should be included, and 3) the influence of higher order corrections even for low fields and small masses. The main idea of these theoretical study and simulations is to ascertain the resolution required in an IMS instrument for any of these effects to be noticeable, which will establish whether or not an empirically observed separation is due to some of these influences, and if it is, to perhaps improve instrument separation by underlining the cause. Preliminary analysis of the equations show that the Mason-Schamp approximation can have up to 4-10% deviations from the exact mobility values at low fields of spherical ions with particular emphasis on very small ions. Part of the deviations may be halved by choosing suitable ion-potential parameters, but this solution is dubiously the most promising way, as the authentic influence of the potentials and higher order effects is obscured. When increasing the field, a shift in mobility starts to be visible even below 8-10 Townsends and can represent up to a deviation of 15-30% for small ions in systems with high field to density ratios. The effect becomes larger for ions with existing dipole moments. The isotopomer separation effect is shown to be tied to differences in the moment of inertia producing shifts in mobility through the speed of rotation of the ion in the gas field. This explanation is supported by experimental results.