Optimized Multi-Attribute Method Workflow Addressing Missed Cleavages and Chromatographic Tailing/Carry-Over of Hydrophobic Peptides

[Image: see text] Peptide mapping by liquid chromatography mass spectrometry (LC-MS) and the related multi-attribute method (MAM) are well-established analytical tools for verification of the primary structure and mapping/quantitation of co- and post-translational modifications (PTMs) or product quality attributes in biopharmaceutical development. Proteolytic digestion is a key step in peptide mapping workflows, which traditionally is labor-intensive, involving multiple manual steps. Recently, simple high-temperature workflows with automatic digestion were introduced, which facilitate robustness and reproducibility across laboratories. Here, a modified workflow with an automatic digestion step is presented, which includes a two-step digestion at high and low temperatures, as opposed to the original one-step digestion at a high temperature. The new automatic digestion workflow significantly reduces the number of missed cleavages, obtaining a more complete digestion profile. In addition, we describe how chromatographic peak tailing and carry-over is dramatically reduced for hydrophobic peptides by switching from the traditional C18 reversed-phase (RP) column chemistry used for peptide mapping to a less retentive C4 column chemistry. No negative impact is observed on MS/MS-derived sequence coverage when switching to a C4 column chemistry. Overall, the new peptide mapping workflow significantly reduces the number of missed cleavages, yielding more robust and simple data interpretation, while providing dramatically reduced tailing and carry-over of hydrophobic peptides.