DOI: 10.1101/497396Dec 16, 2018Paper

Optimising multi-batch TMT analysis to mitigate inflation of missing values, false positives and diminished inter batch accuracy

BioRxiv : the Preprint Server for Biology
Alejandro BrenesAngus I Lamond

Abstract

Multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent, TMT in particular. Here we used a large iPSC proteomic experiment with twenty-four 10-plex TMT batches to evaluate the effect of integrating multiple TMT batches within a single analysis. We reveal a significant inflation rate of missing protein and peptide values and show that precision decreases as multiple batches are integrated. Additionally, we explore the effect of false positives using Y chromosome specific peptides as an internal control to quantify the effect of co-isolation interference, as well as primary and secondary reporter ion interference. Based on the results we suggest solutions to mitigate these effects. We show using a reference line can increase precision by normalising the quantification across batches and we propose experimental designs that minimise the effect of cross population reporter ion interference.

Related Concepts

Cell Separation
Peptides
Evaluation
Genes, Y-Linked
Memory Interference
Research Study
B-Plex
2,5-TMT
Analysis
Thiol S-methyltransferase Activity

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