A "tagless" strategy for identification of stable protein complexes genome-wide by multidimensional orthogonal chromatographic separation and iTRAQ reagent tracking.
Tandem affinity purification is the principal method for purifying and identifying stable protein complexes system-wide in whole cells. Although highly effective, this approach is laborious and impractical in organisms where genetic manipulation is not possible. Here, we propose a novel "tagless" strategy that combines multidimensional separation of endogenous complexes with mass spectrometric monitoring of their composition. In this procedure, putative protein complexes are identified based on the comigration of collections of polypeptides through multiple orthogonal separation steps. We present proof-of-principle evidence for the feasibility of key aspects of this strategy. A majority of Escherichia coli proteins are shown to remain in stable complexes during fractionation of a crude extract through three chromatographic steps. We also demonstrate that iTRAQ reagent-based tracking can quantify relative migration of polypeptides through chromatographic separation media. LC MALDI MS and MS/MS analysis of the iTRAQ-labeled peptides gave reliable relative quantification of 37 components of 13 known E. coli complexes: 95% of known complex components closely co-eluted and 57% were automatically grouped by a prototype computational clustering method. With further technological improvements in each step, we believe this strategy will dramatically improve the efficiency of the purification and identification of protein complexes in cells.
Dong
Ming
M
Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Yang
Lee Lisheng
LL
Williams
Katherine
K
Fisher
Susan J
SJ
Hall
Steven C
SC
Biggin
Mark D
MD
Jin
Jian
J
Witkowska
H Ewa
HE
D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods; D016000:Cluster Analysis; D029968:Escherichia coli Proteins / Q000302:isolation & purification*; D007202:Indicators and Reagents / Q000737:chemistry; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification; D010455:Peptides / Q000302:isolation & purification*; D015203:Reproducibility of Results
D029968:Escherichia coli Proteins; D007202:Indicators and Reagents; D046912:Multiprotein Complexes; D010455:Peptides