Effective correction of experimental errors in quantitative proteomics using stable isotope labeling by amino acids in cell culture (SILAC).

Journal of Proteomics
Sung-Soo ParkStuart Maudsley

Abstract

Accurate and reliable quantitative proteomics in cell culture has been considerably facilitated by the introduction of the stable isotope labeling by amino acids in cell culture (SILAC), combined with high resolution mass spectrometry. There are however several major sources of quantification errors that commonly occur with SILAC techniques, i.e. incomplete incorporation of isotopic amino acids, arginine-to-proline conversion, and experimental errors in final sample mixing. Dataset normalization is a widely adopted solution to such errors, however this may not completely prevent introducing incorrect expression ratios. Here we demonstrate that a label-swap replication of SILAC experiments was able to effectively correct experimental errors by averaging ratios measured in individual replicates using quantitative proteomics and phosphoproteomics of ligand treatment of neural cell cultures. Furthermore, this strategy was successfully applied to a SILAC triplet experiment, which presents a much more complicated experimental matrix, affected by both incomplete labeling and arginine-to-proline conversion. Based on our results, we suggest that SILAC experiments should be designed to incorporate label-swap replications for enhanced rel...Continue Reading

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Citations

Feb 28, 2013·Therapeutic Delivery·Craig RussellAfzal R Mohammed
Apr 20, 2014·Nature Protocols·Sarela García-SantamarinaElena Hidalgo
Jun 13, 2015·Medical Microbiology and Immunology·Arbia KhemiriPascal Cosette
Dec 22, 2015·Journal of Molecular Biology·Caroline M DuffyBrian A Kelch
Jul 15, 2015·Methods : a Companion to Methods in Enzymology·Grace R WilliamsLauren E Ball
May 24, 2015·Journal of the American Society for Mass Spectrometry·Mathieu Lavallée-AdamJohn R Yates
Apr 15, 2015·Nucleic Acids Research·Kshitiz Tyagi, Patrick G A Pedrioli
Feb 18, 2017·Scientific Reports·Christian BorgoLorenzo A Pinna
Sep 27, 2018·PloS One·Alberto ValdésSara Bergström Lind
Dec 22, 2019·International Journal of Molecular Sciences·Fabrizia BrisdelliM Eugenia Schininà
Sep 5, 2020·Scientific Reports·Magdalena ProksovaJiri Stulik
Jul 28, 2019·Scientific Reports·Cristina C SantiniRune Linding
Feb 7, 2021·Molecular Cell·Prashant RawatRitwick Sawarkar
Mar 16, 2021·Molecular & Cellular Proteomics : MCP·Marissa MartinezNatasha E Zachara
Sep 30, 2020·Journal of Proteome Research·Sunkyu ChoiFrank Schmidt
Jun 26, 2021·STAR Protocols·Wankyu LeeUthpala Seneviratne
Jun 12, 2021··Uthpala SeneviratneChristopher W. am Ende

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