Open Access Intact Protein LC-MS in a Recombinant Protein Laboratory
Eric Fang
Thursday, June 19, 2014
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Chapters
Introduction
The Protein Sciences group
Two Key Checkpoints
Failure to perform protein level QC can cause problems!
Rapid intact mass LC/MS of intact antibodies can distinguish them by mass and profile
Rapid intact mass LC/MS of reduced antibodies can distinguish them by primary sequence
Protein average mass measurement is faster and more specific than a Western blot in this environment
A specific primary sequence will have a well defined mass
Example: Fusion protein with post-translational modifications – initial capture step
Example: Mass analysis following dephosphorylation provides additional confirmation
Example: Mass analysis following protease cleavage provides additional confirmation
What is Open Access and why do we want it?
The manual intact protein LC-MS analysis workflow
Automating the intact mass workflow saves time
Automated sequence-based mass matching and annotation potentially saves hundreds of hours per year
Example of an automated protein deconvolution and annotation report
Protein Open Access is a success!
Open Access changes protein MS from a service to a tool
Monitoring of purification fractions
Intact mass can also be useful for proteins and antibodies of unknown sequence
Covalent inhibitor studies
Protein deconvolution results can be used to calculate kinetic parameters
Protein ID by Peptide LC-MS/MS coexists comfortably within our Open Access framework
Open Access to study the effect of compound binding by substrate on its activation by kinase
Studying the effect of substrate compound binding on its phosphorylation by a kinase
Using Heights or Areas can turn qualitative results as on previous slide into (semi-) quantitative ones
Automated annotations can help sift through data
Analysis of peptide LC-MS and LC-MS/MS data can also be automated
Open Access makes Protein Intact Mass more useful
Acknowledgments
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