Mass spectrometry analyses of recombinant hirudins (7 kDa)

The use of liquid secondary ion mass spectrometry (LSIMS) in the characterization of related recombinant 7-kDa peptides illustrates the adequacy of average mass measurement by scanning at low resolution. The difficulty in using the high-resolution technique in the case of poor LSIMS sensitive peptides is discussed, as well as the fact that it does not give, for these molecular weights, any real advantage. The average (or chemical) molecular weights of three recombinant hirudin molecules, hirudin variant 2 (rHV2, 6892.4 Da), hirudin variant 2-Lys47 (rHV2-Lys47, 6906.5 Da), and hirudin variant 2-Arg47 (rHV2-Arg47, 6934.5 Da), less than or equal to 10 micrograms each, have been measured with an accuracy less than or equal to 0.3 Da in the narrow-scan mode and less than or equal to 0.5 Da (from the protonated molecular ion) in the wide-scan mode within 10-15 min; this allows easy distinction of the three 65 amino acid proteins, which differ by a single amino acid. These three molecules could also be distinguished from one another in a mixture. Mass spectrometry and limited sequence characterization of several minor, similarly isolated peptides identified them to be N-terminal additions and/or C-terminal deletions of rHV2-Lys47. LSIMS analysis is consistent with there being no covalent dimer of rHV2-Lys47 as a narrow scan of the 7-kDa molecular ion cluster at high resolution shows it not to be a doubly charged ion.