Health & Environmental Research Online (HERO)


Print Feedback Export to File
1609898 
Journal Article 
Minimizing carry-over in an online pepsin digestion system used for the H/D exchange mass spectrometric analysis of an IgG1 monoclonal antibody 
Majumdar, R; Manikwar, P; Hickey, JM; Arora, J; Middaugh, CR; Volkin, DB; Weis, DD 
2012 
1 
Journal of the American Society for Mass Spectrometry
ISSN: 1044-0305
EISSN: 1879-1123 
23 
12 
2140-2148 
English 
22993047 
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 °C). From a consensus list of 169 different peptides consistently detected during digestion of this large, ~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. 
H/D exchange mass spectrometry; Pepsin; Proteolysis; On-line digestion; Carry-over