Gas-phase concentration, purification, and identification of whole proteins from complex mixtures.
Five proteins present in a relatively complex mixture derived from a whole cell lysate fraction of E. coli have been concentrated, purified, and dissociated in the gas phase, using a quadrupole ion trap mass spectrometer. Concentration of intact protein ions was effected using gas-phase ion/ion proton-transfer reactions in conjunction with mass-to-charge dependent ion "parking" to accumulate protein ions initially dispersed over a range of charge states into a single lower charge state. Sequential ion isolation events interspersed with additional ion parking ion/ion reaction periods were used to "charge-state purify" the protein ion of interest. Five of the most abundant protein components present in the mixture were subjected to this concentration/purification procedure and then dissociated by collisional activation of their intact multiply charged precursor ions. Four of the five proteins were subsequently identified by matching the uninterpreted product ion spectra against a partially annotated protein sequence database, coupled with a novel scoring scheme weighted for the relative abundances of the experimentally observed product ions and the frequency of fragmentations occurring at preferential cleavage sites. The identification of these proteins illustrates the potential of this "top-down" protein identification approach to reduce the reliance on condensed-phase chemistries and extensive separations for complex protein mixture analysis.
Reid
Gavin E
GE
Department of Chemistry, 1393 Brown Building, Purdue University, West Lafayette, Indiana 47907-1393, USA.
Shang
Hao
H
Hogan
Jason M
JM
Lee
Gil U
GU
McLuckey
Scott A
SA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D001426:Bacterial Proteins