Separation and identification of peptides from gel-isolated membrane proteins using a microfabricated device for combined capillary electrophoresis/nanoelectrospray mass spectrometry.
The coupling of microfabricated devices to nanoelectrospray mass spectrometers using both a triple quadrupole and a quadrupole time-of-flight mass spectrometer (QqTOF MS) is presented for the analysis of trace-level membrane proteins. Short disposable nanoelectrospray emitters were directly coupled to the chip device via a low dead volume connection. The analytical performance of this integrated device in terms of sensitivity and reproducibility was evaluated for standard peptide mixtures. A concentration detection limit ranging from 3.2 to 43.5 nM for different peptides was achieved in selected ion monitoring, thus representing a 10-fold improvement in sensitivity compared to that of microelectrospray using the same chip/mass spectrometer. Replicate injections indicated that reproducibility of migration time was typically less than 3.1% RSD whereas RSD values of 6-13% were observed on peak areas. Although complete resolution of individual components is not typically achieved for complex digests, the present chip capillary electrophoresis (chip-CE) device enabled proper sample cleanup and partial separation of multicomponent samples prior to mass spectral identification. Analyses of protein digests were typically achieved in less than 1.5 min with peak widths of 1.8-2.5 s (half-height definition) as indicated from individual reconstructed ion electropherograms. The application of this chip-CE/QqTOF MS system is further demonstrated for the identification of membrane proteins which form a subset of the Haemophilus influenzae proteome. Bands first separated by 1D-gel electrophoresis were excised and digested, and extracted tryptic peptides were loaded on the chip without any further sample cleanup or on-line adsorption preconcentration. Accurate molecular mass determination (< 5 ppm) in peptide-mapping experiments was obtained by introducing an internal standard via a postseparation channel. The analytical potential of this integrated device for the identification of trace-level proteins from different strains of H. influenzae is demonstrated using both peptide mass-fingerprint database searching and on-line tandem mass spectrometry.
Li
J
J
Institute for Biological Sciences, Ottawa, Ontario, Canada.
Kelly
J F
JF
Chernushevich
I
I
Harrison
D J
DJ
Thibault
P
P
D000595:Amino Acid Sequence; D019075:Electrophoresis, Capillary / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006193:Haemophilus influenzae / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
D008565:Membrane Proteins; D010455:Peptides