The analysis of recombinant interleukin-2 by thermospray liquid chromatography-mass spectrometry.
The complete sequence of recombinant human interleukin-2 expressed in Escherichia coli has been confirmed by thermospray liquid chromatography-mass spectrometry (TS-LC-MS) of a tryptic digest derived from 100 micrograms (7 nmol) of reduced carboxymethylated interleukin-2. The preparation was shown by this method to contain predominantly unprocessed N-terminal initiator Met, with some authentic N-terminal Ala; the rest of the protein was as predicted from the DNA sequence, though some deamidated material was noted. TS-LC-MS proved to be a rapid and efficient method for surveying the protein tryptic peptide products allowing all the data to be collected in one chromatographic run; all tryptic fragments were identified by their molecular ions including those for the larger peptides (Mr 1500-3500) which, due to the presence of doubly and triply charged molecular ions, were brought within the mass range of the instrument (1800 Da). It is proposed that TS-LC-MS is a good general method for analyzing recombinant protein digests with respect to sequence confirmation, processing, and post-translational modification, and since each chromatographic peak is identified allows for subsequent monitoring of the protein by LC using uv detection. The method suffers from the disadvantages that all the sample is consumed during the experiment and that no fragment (sequence) ions are generally observed.
Blackstock
W P
WP
Chemical Analysis Department, Glaxo Group Research Ltd., Greenford, Middlesex, United Kingdom.
Dennis
R J
RJ
Lane
S J
SJ
Sparks
J I
JI
Weir
M P
MP
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007376:Interleukin-2; D013058:Mass Spectrometry / Q000379:methods*; D008715:Methionine; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014357:Trypsin
D007376:Interleukin-2; D010446:Peptide Fragments; D011994:Recombinant Proteins; D008715:Methionine; D014357:Trypsin