Multibatch TMT Reveals False Positives, Batch Effects and Missing Values.

Area of Science:

• Proteomics
• Biotechnology
• Mass Spectrometry

Background:

• Multiplexing strategies, particularly Tandem Mass Tag (TMT) labeling, are crucial for large-scale proteomic analyses.
• Integrating multiple TMT batches in a single analysis presents challenges in data quality and interpretation.

Purpose of the Study:

• To evaluate the impact of integrating multiple TMT batches on proteomic data quality.
• To identify and address limitations in multi-batch TMT analyses, including missing values, false positives, and batch effects.

Main Methods:

• Utilized a large induced pluripotent stem cell (iPSC) proteomic experiment with twenty-four 10-plex TMT batches.
• Employed Y chromosome peptides as internal controls to assess false positives and ion co-isolation.
• Investigated reporter ion interference and proposed normalization strategies.

Main Results:

• Observed a significant inflation of protein and peptide missing values with increasing numbers of integrated TMT batches.
• Detected Y chromosome-specific peptides in female samples, indicating false positives and ion interference.
• Demonstrated that without normalization, the precision of single-batch TMT analysis is not maintained across multiple batches.

Conclusions:

• Multi-batch TMT analyses require robust normalization strategies to mitigate batch effects and improve data reliability.
• Including a common reference line in each batch enhances precision and facilitates cross-batch normalization.
• Proposed experimental designs to minimize reporter ion interference and improve the accuracy of large-scale proteomic studies.