Quantitative proteomics reveals significant changes in cell shape and an energy shift after IPTG induction via an optimized SILAC approach for Escherichia coli.
Stable isotope labeling by amino acids in cell culture (SILAC) has been widely used in yeast, mammalian cells, and even some multicellular organisms. However, the lack of optimized SILAC media limits its application in Escherichia coli, the most commonly used model organism. We optimized SILACE medium (SILAC medium created in this study for E. coli) for nonauxotrophic E. coli with high growth speed and complete labeling efficiency of the whole proteome in 12 generations. We applied a swapped SILAC workflow and pure null experiment with the SILACE medium using E. coli BL21 (DE3) cells hosting a recombinant plasmid coding for glutathione-S-transferase (GST) and ubiquitin binding domain before and after isopropyl thiogalactoside (IPTG) induction. Finally, we identified 1251 proteins with a significant change in abundance. Pathway analysis suggested that cell growth and fissiparism were inhibited accompanied by the down-regulation of proteins related to energy and metabolism, cell division, and the cell cycle, resulting in the size and shape change of the induced cells. Taken together, the results confirm the development of SILACE medium suitable for efficient and complete labeling of E. coli cells and a data filtering strategy for SILAC-based quantitative proteomics studies of E. coli.
State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine , Beijing 102206, P. R. China.
Lingyan
L
Ping
Heng
H
Zhang
Linhui
L
Zhai
Eric B
EB
Dammer
Duc M
DM
Duong
Ning
N
Li
Zili
Z
Yan
Junzhu
J
Wu
Ping
P
Xu
D002247:Carbon Isotopes; D002455:Cell Division; D003470:Culture Media / Q000737:chemistry; D004734:Energy Metabolism / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology*; D007553:Isotope Labeling / Q000379:methods*; D010957:Plasmids; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*; D015533:Transcriptional Activation; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism
D002247:Carbon Isotopes; D003470:Culture Media; D029968:Escherichia coli Proteins; D025801:Ubiquitin; D007544:Isopropyl Thiogalactoside; D005982:Glutathione Transferase