TY - JOUR
T1 - Simultaneous Analysis of a Non-Lipidated Protein and Its Lipidated Counterpart
T2 - Enabling Quantitative Investigation of Protein Lipidation's Impact on Cellular Regulation
AU - Shala-Lawrence, Agnesa
AU - Blanden, Melanie J.
AU - Krylova, Svetlana M.
AU - Gangopadhyay, Soumyashree A.
AU - Beloborodov, Stanislav S.
AU - Hougland, James L.
AU - Krylov, Sergey N.
N1 - Publisher Copyright:
© 2017 American Chemical Society.
PY - 2017/12/19
Y1 - 2017/12/19
N2 - Here, we introduce protein-lipidation quantitation (PLQ) - the first method for quantitative analysis of both a substrate and a product of protein lipidation in a biologically relevant context. Such analysis is required to study roles of protein lipidation in cellular regulation. In PLQ, the substrate is fused with a fluorescent protein to facilitate quantitative detection of both the nonlipidated substrate and the lipidated product. When expressed in cells with endogenous lipidation activity, the substrate is intracellularly lipidated. Following cell lysis and sampling crude cell lysate for analysis, the substrate and the product are separated by surfactant-mediated capillary electrophoresis (CE) and quantitated by integrating fluorescence intensity over respective CE peaks. In this work, we prove PLQ in principle and demonstrate its robustness to changes in structures of the substrate and lipid donor. Finally, PLQ analysis confirms a hypothesized link between a mutation in p53 and cellular prenylation activity.
AB - Here, we introduce protein-lipidation quantitation (PLQ) - the first method for quantitative analysis of both a substrate and a product of protein lipidation in a biologically relevant context. Such analysis is required to study roles of protein lipidation in cellular regulation. In PLQ, the substrate is fused with a fluorescent protein to facilitate quantitative detection of both the nonlipidated substrate and the lipidated product. When expressed in cells with endogenous lipidation activity, the substrate is intracellularly lipidated. Following cell lysis and sampling crude cell lysate for analysis, the substrate and the product are separated by surfactant-mediated capillary electrophoresis (CE) and quantitated by integrating fluorescence intensity over respective CE peaks. In this work, we prove PLQ in principle and demonstrate its robustness to changes in structures of the substrate and lipid donor. Finally, PLQ analysis confirms a hypothesized link between a mutation in p53 and cellular prenylation activity.
UR - http://www.scopus.com/inward/record.url?scp=85038863644&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85038863644&partnerID=8YFLogxK
U2 - 10.1021/acs.analchem.7b03846
DO - 10.1021/acs.analchem.7b03846
M3 - Article
C2 - 29144728
AN - SCOPUS:85038863644
SN - 0003-2700
VL - 89
SP - 13502
EP - 13507
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 24
ER -