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Buck Institute for Research on Aging

9 ARTICLES PUBLISHED IN JoVE

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Biology

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples
Eric Johansen 1, Birgit Schilling 2, Michael Lerch 1, Richard K. Niles 1, Haichuan Liu 1, Bensheng Li 2, Simon Allen 1, Steven C. Hall 1, H. Ewa Witkowska 1, Fred E. Regnier 3, Bradford W. Gibson 2, Susan J. Fisher 1, Penelope M. Drake 1
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.

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Biology

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes
James M. Flynn 1, Luis F. Santana 2, Simon Melov 1
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.

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Biology

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate
Shona A. Mookerjee 1,2, Martin D. Brand 1,2
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.

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Biochemistry

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans
Mark Lucanic 1, Theo Garrett 1, Matthew S. Gill 2, Gordon J. Lithgow 1
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.

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JoVE Core

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry
Lei Wei 1, Jesse G. Meyer 1, Birgit Schilling 1
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.

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Genetics

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis
Carly V. Weiss 1,2, Julie N. Chuong 3, Rachel B. Brem 1,3
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.

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Biology

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization
Xueshu Xie 1, Samah Shah 1, Anja Holtz 1, Jacob Rose 1, Nathan Basisty 1, Birgit Schilling 1
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.

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Biology

Quantification of Insoluble Protein Aggregation in Caenorhabditis elegans during Aging with a Novel Data-Independent Acquisition Workflow
Xueshu Xie *1, Manish Chamoli *1, Dipa Bhaumik 1, Renuka Sivapatham 1, Suzanne Angeli 1, Julie K. Andersen 1, Gordon J. Lithgow 1, Birgit Schilling 1
1Buck Institute for Research on Aging

This novel workflow efficiently extracts and isolates SDS-insoluble proteins (insolublome) from Caenorhabditis elegans with minimal starting material for quantitative differential proteomic analysis. The protocol uses a comprehensive data-independent acquisition mass spectrometry analysis to quantify the insolublome and bioinformatic analysis to gain biological insights into aging mechanisms and pathologies.

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Biochemistry

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry
Matthew Payea 1, Myriam Gorospe 1, Nathan Basisty 2
1Laboratory of Genetics and Genomics, National Institute on Aging Intramural Research Program, National Institutes of Health, 2Translational Gerontology Branch, National Institute on Aging Intramural Research Program, National Institutes of Health

This protocol describes the workflow for metabolic labeling of senescent and non-dividing cells with pulsed SILAC, untargeted mass spectrometry analysis, and a streamlined calculation of protein half-lives.

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