In Host Cell Protein (HCP) analysis, the number of identified proteins can vary depending on whether a sample is analyzed individually or as part of a batch with other samples. This behavior is specific to HCP workflows and is influenced by the Auto-decoy setting used during analysis. A lower Auto-decoy value can lead to noticeable differences in protein identification (see the referenced article for more details).
Figure 1: Individual Sample Analysis with Auto-decoy = 2
In this scenario, each sample is analyzed separately with the Auto-decoy setting set to 2. Proteins are included only if they rank above the third decoy. For Sample A, this results in three protein hits. For Sample B, four protein hits are identified. Although Sample A also detected 'Prot 4', it was excluded because its rank was below 'Decoy 3'.
Figure 2: Combined Sample Analysis with Auto-decoy = 2
Here, both samples are analyzed together using the same Auto-decoy setting. Since 'Prot 4' is included for Sample B (as it ranks above the third decoy in the combined analysis), it is also listed for Sample A—even though it would have been excluded if Sample A were analyzed alone.
Conclusion
When multiple samples are analyzed together, any protein that surpasses the Auto-decoy threshold in one sample will be considered across all samples in the analysis. This can result in proteins being reported in samples where they would otherwise fall below the threshold if analyzed individually.
To avoid this effect and ensure consistent protein identification regardless of whether samples are run individually or in a batch, set Auto-decoy = 100. With this setting, the number of protein hits will remain stable across both single-sample and multi-sample analyses.