Buck Institute for Research on Aging View Institution's Website 8 articles published in JoVE Biology Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization Xueshu Xie1, Samah Shah1, Anja Holtz1, Jacob Rose1, Nathan Basisty1, Birgit Schilling1 1Buck Institute for Research on Aging This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk. Genetics Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis Carly V. Weiss1,2, Julie N. Chuong3, Rachel B. Brem1,3 1Department of Plant and Microbial Biology, University of California Berkeley, 2Department of Biology, Stanford University, 3Buck Institute for Research on Aging Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait. Biology Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry Lei Wei1, Jesse G. Meyer1, Birgit Schilling1 1Buck Institute for Research on Aging Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained. Biochemistry A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans Mark Lucanic1, Theo Garrett1, Matthew S. Gill2, Gordon J. Lithgow1 1The Buck Institute for Research on Aging, 2The Scripps Research Institute Center on Aging Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects. Biology Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate Shona A. Mookerjee1,2, Martin D. Brand1,2 1Touro University California College of Pharmacy, 2Buck Institute for Research on Aging Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification. Biology Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes James M. Flynn1, Luis F. Santana2, Simon Melov1 1Buck Institute for Research on Aging, 2Department of Physiology & Biophysics, University of Washington Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets. Biology Bioenergetic Profile Experiment using C2C12 Myoblast Cells David G. Nicholls1, Victor M. Darley-Usmar2, Min Wu3, Per Bo Jensen3, George W. Rogers3, David A. Ferrick3 1Buck Institute for Age Research, Novato, CA, 2Department of Pathology, Center for Free Radical Biology, University of Alabama at Birmingham - UAB, 3Seahorse Bioscience, North Billerica, MA A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity. Biology A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples Eric Johansen1, Birgit Schilling2, Michael Lerch1, Richard K. Niles1, Haichuan Liu1, Bensheng Li2, Simon Allen1, Steven C. Hall1, H. Ewa Witkowska1, Fred E. Regnier3, Bradford W. Gibson2, Susan J. Fisher1, Penelope M. Drake1 1Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco - UCSF, 2Buck Institute for Age Research, 3Department of Chemistry, Purdue University Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.
