Integration of electrokinetic-based multidimensional separation/concentration platform with electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry for proteome analysis of Shewanella oneidensis.
This work focuses on the development of a multidimensional electrokinetic-based separation/concentration platform coupled with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) for achieving the high resolution and ultrasensitive analysis of complex protein/peptide mixtures. A microdialysis junction is employed as the interface for on-line combination of capillary isoelectric focusing (CIEF) with transient capillary isotachophoresis/zone electrophoresis (CITP/CZE) in an integrated platform. Besides the excellent resolving power afforded by both CIEF and CZE separations, the electrokinetic focusing/stacking effects of CIEF and CITP greatly enhance the dynamic range and detection sensitivity of MS for protein identification. The constructed multidimensional separation/concentration platform is demonstrated for the analysis of Shewanella oneidensis proteome, which has considerable implications toward the bioremediation of environmental pollutants. The electrokinetic-based platform offers the overall peak capacity comparable to those obtained using multidimensional chromatography systems, but with a much shorter run time and no need for column regeneration. Most importantly, a total of 1174 unique proteins, corresponding to 26.5% proteome coverage, are identified from the cytosolic fraction of S. oneidensis, while requiring <500 ng of proteolytic digest loaded in the CIEF capillary. The ultrasensitive capabilities of electrokinetic-based proteome approach are attributed to the concentration effect in CIEF, the electrokinetic stacking of CITP, the nanoscale peak volume in CZE, the "accurate mass tag" strategy for protein/peptide identification, and the high-sensitivity, high-resolution, and high-mass measurement accuracy of FTICR-MS.
Mohan
Deepa
D
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA.
Pasa-Tolić
Ljiljana
L
Masselon
Christophe D
CD
Tolić
Nikola
N
Bogdanov
Bogdan
B
Hixson
Kim K
KK
Smith
Richard D
RD
Lee
Cheng S
CS
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis*; D002417:Cattle; D017357:Cyclotrons; D004563:Electrochemistry; D019075:Electrophoresis, Capillary; D015180:Electrophoresis, Gel, Two-Dimensional; D005583:Fourier Analysis; D007525:Isoelectric Focusing; D007700:Kinetics; D008969:Molecular Sequence Data; D020543:Proteome / Q000032:analysis*; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000378:metabolism; D020592:Shewanella / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation* / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D001426:Bacterial Proteins; D020543:Proteome; D012710:Serum Albumin, Bovine; D014357:Trypsin