Bombesin, separation

The basic polymer appears to be a hydroxylated polyether to which octadecyl chains have been bonded and so it behaves as a reverse phase exhibiting dispersive interactions with the solutes. An example of the separation of a series of peptides is shown in figure 15. The column was 3.5 cm long, 4.6 mm i.d. The solutes shown were (1) oc-endorphin, (2) bombesin, (3) y-endorphin, (4) angiotensin, (5) somatostatin and (6) calcitonon. The separation was carried out with a 10 min linear program from water containing 0.2% trifluoroacetic acid to 80% acetonitrile. 

Figure 5.2 Effect of ionic strength on the separation of bioactive peptides. Conditions capillary, 375 gm O.D. X 75 /u.m I.D. x 57 cm (50 cm to detector) separation voltage, 30 kV temperature, 30°C buffer, 0.025 M-0.125 M sodium dihydrogen phosphate, pH 2.44. (Reprinted from Ref. 5 with permission.) (1) Bradykinin (BRAD) (2) Angiotensin II(ANG II) (3) Thyrotropin releasing hormone (TRH) (4) Luteinizing hormone releasing hormone (LHRH) (5) Bombesin (BOMB) (6) Leucine enkephalin (LENK) (7) Methionine enkephalin (MENK) (8) Oxytocin (OXYT) (9) Dynorphin (DYNO).

Fig. 5. The separation of polypeptide standards on Hypersil ODS with 0.1 M NaHjP04-H3P04, pH 2.1, as the mobile phase, at a flow rate of 1 ml/min. The peaks are as follows 1, Trp 2, lysine vasopressin 3, arginine vasopressin 4, oxytocin, 5, ACTHj-j, 6, insulin A-chain 7, bombesin 8, substance P 9, somatostatin 10, insulin B-chain 11, human calcitonin 12, glucagon 13, salmon calcitonin 14, melittin. Adapted from Fig. I of O Hare and Nice (1979).

Fic. 11. Separation of bombesin (l)from [D-Met,4]-bombesin(2)by HPLC. Conditions -Bondapak Cn column (120 x 0.7 cm) flow rate 6 ml/min isocratic elution using 30% acetonitrile-10 mAf ammonium acetate, pH 4.2. Reprinted with permission from Rivier and Brown 49). Copyright by American Chemical Society. 

Figure 10.6 Upper panel shows a two-dimensional separation contour plot showing the separation of peptides bradykinin, substance p and bombesin on a PMMA chip. The x axis represents m/z values, the y axis represents the separation and readout times and the gray shade shows the ion intensities. The panel below is a representative electropherogram generated by integrating ion intensities from a two-dimensional plot.

Figure 5.1-2. Electropherogram of a mixture of bioactive peptides separated at pH 2.5. Peptides (1) reference (2) bradykinin (3) bradykinin fragment 1-5 (4)substance P (5) [arg]-vasopressin (6) luteinizing hormone releasing hormone (7) bombesin (8) leucine enkephalin (9) methionine enkephalin (10) oxytocin. (Reprinted from Ref. 13, with permission.)...

Pettitt, TR and Wakelam, MJ (1993) Bombesin stimulates distinct time-dependent changes in the sn-l,2-diradylglycerol molecular species profile from Swiss 3T3 fibroblasts as analysed by 3,5-dinitrobenzoyl derivatization and h.p.l.c. separation. Biochemical Journal, 289, 487 95. 

When more than one peptide is present, ion gating is required as shown in Figure 8.11. In these spectra, obtained at fvill reflectron voltage, both focused and small PSD fragments are observed for each component, but can be separated by the application of an ion gate. Figure 8.12 shows the assembled product ion mass spectrum of bombesin obtained from this mixture from 12 mass-spectral segments. 

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