PLIMSTEX assays give H/D exchange profiles that provide a global view of the intact protein. One of the advantages of using MS to measure exchange is that the information can be extended to the peptide and even the amino-acid level by enzyme digestion and/or by MS/MS analysis [102-107]. Once the binding affinity and protection in the intact protein are determined by PLIMSTEX, the resolution of the information can be increased by digesting the protein with pepsin after the exchange is quenched (pepsin works at the low pH of the quench). The resulting peptides can be analyzed by MALDI-MS, or LC/ESI-MS and MS/MS. We compared different approaches for pepsin digestion  of IFABP, CaM, and ras protein. The online digestion on a custom-built immobilized pepsin column [53, 108] followed by LC-MS and MS/MS may give the best sequence coverage and experimental control. Compared to a solution approach, there is less pepsin interference in the mass spectrum, more complete digestion, more reproducible cleavage sites, and less digestion time (leading to less back exchange). We applied this online digestion to ligand binding of IFABP .
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