Protein digest analysis by pressurized capillary electrochromatography using an ion trap storage/reflectron time-of-flight mass detector.
Pressurized capillary electrochromatography (pCEC) has been coupled to an ion trap storage/reflectron time-of-flight mass spectrometer for the analysis of peptide mixtures and protein digests. Taking advantage of the electroosmotic flow, high separation efficiency has been achieved in pCEC due to a relatively flat flow profile and the use of smaller packing materials. A supplementary pressure was used in these experiments which suppressed bubble formation and also allowed the tuning of the elution of peptides using the electrical field. In this work, a fast separation of a six-peptide mixture has been successfully performed. Using columns only 6 cm long, a tryptic digest of bovine cytochrome c was fully separated in around 14 min by properly tuning the applied voltage and the supplementary pressure. In addition, relatively complex protein digests, such as a tryptic digest of chicken ovalbumin, were analyzed using this pCEC/MS system, and more than 20 peaks were resolved in the total ion current chromatogram within 17 min. The use of an ion trap storage/reflectron time-of-flight mass spectrometer as an on-line detector further increased the resolving power of the pCEC by unambiguously identifying coeluting components. The nonscanning property of the time-of flight mass analyzer and the ion signal integration capability of the ion trap were successfully combined to provide rapid and sensitive full-mass range detection in these experiments.
Wu
J T
JT
Department of Chemistry, University of Michigan, Ann Arbor 48109, USA.
Huang
P
P
Li
M X
MX
Lubman
D M
DM
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002645:Chickens; D003574:Cytochrome c Group / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D010449:Peptide Mapping; D011312:Pressure; D014357:Trypsin / Q000737:chemistry
D003574:Cytochrome c Group; D010047:Ovalbumin; D014357:Trypsin