Peptides bovine serum albumin, tryptic digest

Fig. 11.6. Peptide sequencing by nanoESI-CID-MS/MS from a tryptic digest of bovine serum albumin (BSA) 800 fmol of BSA were used, (a) Eull scan spectrum, (b) fragmentation of the selected doubly charged peptide ion at m/z 740.5. Adapted from Ref. [66] by permission. Nature Publishing Group, 1996.

FIGURE 4.13 A MALDI-TOF mass spectrum acquired in reflectron mode showing the tryptic peptides from a digestion of bovine serum albumin. The m/z of the monoisotopic ions are assigned and can be entered into a protein database search engine to match to the theoretical trypsin digest of BSA. 

The spectrophotometric evidence reviewed above for the binding of a proportion of the phenolic hydroxyl groups of the tyrosine residues of native proteins is supported by work on the action of tyrosinase on proteins. Sizer (1946) found that this enzyme oxidizes the tyrosine residues in native trypsin, pepsin, chymotrypsin, casein, peptone, insulin, and hemoglobin. Native ovalbumin, human and bovine serum albumin, tobacco mosaic virus (nucleoprotein), human y- and bovine /3-globulins, and bovine fibrinogen are not susceptible to tyrosinase, but become so after tryptic digestion. It was shown (Sizer, 1947) that for the proteins which are oxidized by tyrosinase in the native state, the observed reaction does indeed occur with the intact proteins and does not require preliminary degradation to tyrosine peptides or free tyrosine. The kinetics of the oxidation of tyrosine by tyrosinase have been studied spectropho-tometrically (Mason, 1948 etc.). 

The performance of the device was demonstrated with the separation of a tryptic digest of bovine serum albumin (BS A) in the reversed-phase mode using a gradient of acetonitrile in aqueous solution of trifiuoroacetic acid as the mobile phase. The separated peptides were detected by mass spectrometer. Figure 47.6 shows both the device and separation of a sample containing 5 pmol of peptides. Comparison with a database revealed an excellent amino acid sequence coverage of 70%. 

Example DIOS-TOF/TOF experiments on the tryptic digest of 100 fmol of the protein bovine serum albumine (BSA) demonstrate this technique. Using conductive tape the DIOS chip was directly attached to the MALDI target plate of an Applied Biosystems 4700 tandem TOF instrument equipped with a frequency-tripled Nd YAG laser (355 nm). The resulting DIOS-TOF spectmm of the digest and the DIOS-TOF/TOF spectrum of one selected peptide, [YLYEIAR+H]", m/z 927.5 are shown in Fig. 11.27. The tandem mass spectrum provides complete detection of the y and b ion series [192]. 

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