Trypsin hydrolysis/HPLC/thermospray analysis

Figure 2. A) Trypsin hydrolysis/HPLC/thermospray MS analysis of Y-endorphin. The two tryptic fragments (T1 and T ) were separated by HPLC (10% isopropanol in aqueous ammonium acetate (0.1 M) pH 7.1, 1 mL/min) and detected by thermospray MS. B,B ) Thermospray mass spectra of tryptic fragments and Tg separated by HPLC after hydrolysis. C,Cj) Thermospray spectra from trypsin hydrolysis followed by HPLC then carboxypeptidase A, B, and Y hydrolysis for the partial amino acid sequencing of Y-endorphin.

The combination of immobilized enzyme columns with HPLC/thermospray MS can be very useful in peptide identification and sequencing [6,7], There are a number of ways of combining the immobilized enzyme column, HPLC and MS detection for peptide analysis. Use of an endopeptidase column prior to HPI.C separation and MS detection will enable separation of each hydrolysis product for Identification. Figure 2 shows the trypsin column/HPLC/thermospray MS of Y-endorphin. The selected ion chromatograms show the retention time for each tryptic hydrolysis product Tj and T. Typically, this column configuration can only be used on purified samples since no separation or column clean-up is performed before hydrolysis. 

Combination of two immobilized enzyme columns with HPLC/thermospray MS can be useful for amino acid sequencing and identification. The use of an endopeptidase bioreactor followed by HPLC separation then an exopeptidase column and MS detection can enable sequencing of 3-5 amino acids of each endopeptidase hydrolysis product. The trypsin, hydrolysis/HPLC/ carboxypeptidase A, B, and Y (1 1 1) hydrolysis/ thermospray MS analysis assist in the sequencing of Y-endorphin (Figure 2C,C ). 

Figure 3. HPLC/trypsin hydrolysis/thermospray MS analysis of a mixture of two endorphins in a plasma extract. The separation was performed using 25% isopropanol in aqueous ammonium acetate (0.1 H) solution at a pH of 7.5 at a flow of 1.0 mL/min.

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See also in sourсe #XX -- [ , , , ]

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