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

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.

Automated summary

Peptide mapping by LC-MS and the related MAM are commonly used analytical tools in biopharmaceutical development for verifying protein structure and mapping/quantifying modifications or product quality attributes. Traditional proteolytic digestion workflows are labor-intensive, but recently introduced high-temperature automatic workflows have improved robustness and reproducibility. This article presents a modified automatic digestion workflow involving two-step digestion at high and low temperatures, which reduces missed cleavages and improves digestion completeness. Switching from traditional C18 to less retentive C4 column chemistry also reduces peak tailing and carry-over of hydrophobic peptides without compromising MS/MS-derived sequence coverage.