Multibatch TMT reveals false positives, batch effects and missing values
Open accessAbstract
Multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent, tandem mass tags (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 identified a significant inflation rate of protein missing values as multiple batches are integrated and show that this pattern is aggravated at the peptide level. We also show that without normalization strategies to address the batch effects, the high precision of quantitation within a single multiplexed TMT batch is not reproduced when data from multiple TMT batches are integrated.Further, the incidence of false positives was studied by using Y chromosome peptides as an internal control. The iPSC lines quantified in this data set were derived from both male and female donors, hence the peptides mapped to the Y chromosome should be absent from female lines. Nonetheless, these Y chromosome-specific peptides were consistently detected in the female channels of all TMT batches. We then used the same Y chromosome specific peptides to quantify the level of ion coisolation as well as the effect of primary and secondary reporter ion interference. These results were used to propose solutions to mitigate the limitations of multi-batch TMT analyses. We confirm that including a common reference line in every batch increases precision by facilitating normalization across the batches and we propose experimental designs that minimize the effect of cross population reporter ion interference.
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It is not uncommon to have 20-50% of the possible peptide values that are not quantified.
“It is not uncommon to have 20–50% of the possible peptide values that are not quantified (Webb-Robertson et al., 2015; Brenes et al., 2019).”
From Missing data in multi-omics integration: Recent advances through artificial intelligence (2023) · cites this paper as Brenes et al., 2019
Multiplexing methods are limited in how many samples can be tagged and analyzed together, and the same peptides are sampled less frequently across batches than proteins are.
“These multiplexing methods are still limited in the number of samples that can be uniquely tagged, combined, and analyzed at once, and while multiple batches of samples can be acquired, the same peptides are less frequently sampled in different batches compared to proteins.(9)”
From Putting humpty dumpty back together again: what does protein quantification mean in bottom-up proteomics? (2022) · cites this paper as [9]
TMT-MS may still yield missing values across multiplexed batches when analyzing large numbers of samples.
“However, despite these achievements in quantification, TMT-MS may still yield missing values across multiplexed batches when analyzing large numbers of samples [80].”
From Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau (2021) · cites this paper as [80]
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“It is not uncommon to have 20–50% of the possible peptide values that are not quantified (WebbRobertson et al., 2015; Brenes et al., 2019).”
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- Authors
- Brenes, A., Hukelmann, J., Bensaddek, D., & Lamond, A. I.
- Affiliations
- University of Dundee (GB)
- Corresponding author
- Lamond, Angus I.
- Venue
- Molecular & Cellular Proteomics
- Publisher
- Elsevier BV
- Published
- 2019-10
- Volume
- 18
- Issue
- 10
- Pages
- 1967-1980
- ISSN
- 1535-9476 (ISSN-L)
- PMC
- PMC6773557
- OpenAlex
- W2963597305
- Cited by
- 196
- References
- 43
- Citations by year
- 2026: 17 · 2025: 31 · 2024: 28 · 2023: 37 · 2022: 23 · 2021: 31 · 2020: 26 · 2019: 2 · 2018: 1
- FWCI
- 8.19
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Molecular & Cellular Proteomics (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Discovery Research Portal (University of Dundee) (publishedVersion, cc-by)
- Edinburgh Research Explorer (University of Edinburgh) (submittedVersion, cc-by)
- King Abdullah University of Science and Technology Repository (King Abdullah University of Science and Technology) (submittedVersion)
- Funders
- UK Research and Innovation | Medical Research Council (098503/E/12/Z)
- Wellcome Trust (098503/E/12/Z, 073980/Z/03/Z, 105024/Z/14/Z)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced Biosensing Techniques and Applications
- Keywords
- tandem mass tags, batch effects, false positives, induced pluripotent stem cells, data normalization, peptide quantification, multiplexed proteomics
- MeSH terms
- Cells, Cultured, Chromatography, Liquid, Female, Humans, Male, Peptides, Reproducibility of Results, Proteomics, Chromosomes, Human, Y, Tandem Mass Spectrometry, Induced Pluripotent Stem Cells
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