In the FT-ICR approach to protein identification, both “top down” (fragmentation of the intact protein mass) or “bottom-up” (MS analysis of enzymatic digests) analyses are performed on a Bruker Apex Qe 9.4T FT-ICR instrument. This instrument has three methods for fragmentation of peptides/proteins: SORI-CAD (sustained off-resonance irradiation – collisionally activated dissociation), InfraRed MultiPhoton Dissociation (IRMPD), and ECD (Electron Capture Dissociation).
For intact protein identification (< ~ 35kDa in size), ECD or a combination of ECD and IRMPD is typically used to fragment the protein of interest. These fragment ions are measured at a high mass accuracy (<3ppm) to allow for accurate identification of the protein. By using the MS/MS methods individually or in conjunction with each other, we are able to identify a higher percentage of the intact sequence to be identified as compared to using the conventional approach of a tryptic digest and MS/MS. In the tryptic approach, we are limited by the number of cleavage sites (after Lys and Arg) while in ECD, IRMPD and CAD, any peptide bond can theoretically cleave (1). For proteins in the 20-45 kDa mass range the mass measurement error is usually ±1Da (1)
The FT-ICR bottom-up analysis is performed on enzymatic digests, often without prior fractionation. The main advantage of this approach over a typical LC-MS/MS analysis is the very high mass accuracy of the FT-ICR. Figure 1 shows the mass spectrum of an unfractionated tryptic digest mixture of four proteins. The tryptic digest mixture was desalted with a C18 ZipTip and eluted into 60% methanol containing 0.1% acetic acid. This was then electrosprayed into the FT-ICR for accurate mass measurements. The accurate masses of the peaks were used in the database search for protein identifications and the 4 proteins (BSA, carbonic anhydrase, myoglobin, and alcohol dehydrogenase) were identified. Protein identifications are typically validated by MS/MS fragmentation from any one of the fragmentation techniques of FT-ICR MS (CID, IRMPD, and ECD).
- Shi, S.D. Hemling, M.E., Carr, S.A., Horn, D.M., Lindh, I. and McLafferty, F.W., (2001) Phosphopeptide/phosphoprotein mapping by electron capture dissociation mass spectrometry. Anal. Chem. 73, 19-22.