Figure 6 (left) HPLC mass chromatogram for the tryptic digest of ovalbumin. (right) The resulting mass spectra at the indicated retention times are shown. Approximately 0.25 µg of protein was loaded onto an SDS-PAGE gel, and the protein was stained with zinc imidazole. An electrospray ionization ion trap mass spectrometer (LCQ, Finnigan MAT, San Jose, CA) coupled on-line with a capillary HPLC (MicroPro Syringe Pumping System, Eldex Laboratories, Napa, CA) injunction with a capillary peptide trapping precolumn was used for peptide analysis.

doi:10.1201/9780203910696-20

Chapter

Figure 6 (left) HPLC mass chromatogram for the tryptic digest of ovalbumin. (right) The resulting mass spectra at the indicated retention times are shown. Approximately 0.25 µg of protein was loaded onto an SDS-PAGE gel, and the protein was stained with zinc imidazole. An electrospray ionization ion trap mass spectrometer (LCQ, Finnigan MAT, San Jose, CA) coupled on-line with a capillary HPLC (MicroPro Syringe Pumping System, Eldex Laboratories, Napa, CA) injunction with a capillary peptide trapping precolumn was used for peptide analysis.

Chapter

Figure 6 (left) HPLC mass chromatogram for the tryptic digest of ovalbumin. (right) The resulting mass spectra at the indicated retention times are shown. Approximately 0.25 µg of protein was loaded onto an SDS-PAGE gel, and the protein was stained with zinc imidazole. An electrospray ionization ion trap mass spectrometer (LCQ, Finnigan MAT, San Jose, CA) coupled on-line with a capillary HPLC (MicroPro Syringe Pumping System, Eldex Laboratories, Napa, CA) injunction with a capillary peptide trapping precolumn was used for peptide analysis.

ABSTRACT

For a single sample, LC-MS/MS analysis included two discrete steps: (1) LC-MS peptide mapping to identify peptide ions from the digestion mixture and to deduce their molecular weights, and (2) LC-MS/MS of the previously detected peptides to obtain sequence information for protein identification. An improvement in efficiency and throughput of the overall method can be obtained by performing LC-MS/MS in the datadependent mode. As full-scan mass spectra are acquired continuously in LC-MS mode, any ion detected with a signal intensity above a predefined threshold will trigger the mass spectrometer to switch to MS/MS mode. Thus, the ion trap mass spectrometer switches back and forth between MS (molecular mass information) and MS/MS mode (sequence information) in a single LC run. This feature was implemented to generate the spectra shown in Figure 7. The data-dependent scanning capability, combined with an autosampler device, can dramatically increase the capacity and throughput for protein identification.