期刊
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
卷 23, 期 12, 页码 2140-2148出版社
SPRINGER
DOI: 10.1007/s13361-012-0485-9
关键词
H/D exchange mass spectrometry; Pepsin; Proteolysis; On-line digestion; Carry-over
资金
- MedImmune, LLC.
Chromatographic carry-over can severely distort measurements of amide H/D exchange in proteins analyzed by LC/MS. In this work, we explored the origin of carry-over in the online digestion of an IgG1 monoclonal antibody using an immobilized pepsin column under quenched H/D exchange conditions (pH 2.5, 0 A degrees C). From a consensus list of 169 different peptides consistently detected during digestion of this large, similar to 150 kDa protein, approximately 30 % of the peptic peptides exhibited carry-over. The majority of carry-over originates from the online digestion. Carry-over can be substantially decreased by washing the online digestion flow-path and pepsin column with two wash cocktails: [acetonitrile (5 %)/ isopropanol (5 %)/ acetic acid (20 %) in water] and [2 M guanidine hydrochloride in 100 mM phosphate buffer pH 2.5]. Extended use of this two-step washing procedure does not adversely affect the specificity or activity of the immobilized pepsin column. The results suggest that although the mechanism of carry-over appears to be chemical in nature, and not hydrodynamic, carry-over cannot be attributed to a single factor such as mass, abundance, pI, or hydrophobicity of the peptides.
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