Accedi

Buck Institute for Age Research

3 ARTICLES PUBLISHED IN JoVE

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.

JoVE Core

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.

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.