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Published on: November 15, 2017
TMTpro Complementary Ion Quantification Increases Plexing and Sensitivity for Accurate Multiplexed Proteomics at the
Alex Johnson1,2,3, Michael Stadlmeier1,2, Martin Wühr1,2,3
1Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, United States.
Abstract:
Multiplexed proteomics is a powerful tool to assay cell states in health and disease, but accurate quantification of relative protein changes is impaired by interference from co-isolated peptides. Interference can be reduced by using MS3-based quantification, but this reduces sensitivity and requires specialized instrumentation. An alternative approach is quantification by complementary ions, the balancer group-peptide conjugates, which allows accurate and precise multiplexed quantification at the MS2 level and is compatible with most proteomics instruments. However, complementary ions of the popular TMT-tag form inefficiently and multiplexing is limited to five channels. Here, we evaluate and optimize complementary ion quantification for the recently released TMTpro-tag, which increases complementary ion plexing capacity to eight channels (TMTproC). Furthermore, the beneficial fragmentation properties of TMTpro increase sensitivity for TMTproC, resulting in ∼65% more proteins quantified compared to TMTpro-MS3 and ∼18% more when compared to real-time-search TMTpro-MS3 (RTS-SPS-MS3). TMTproC quantification is more accurate than TMTpro-MS2 and even superior to RTS-SPS-MS3. We provide the software for quantifying TMTproC data as an executable that is compatible with the MaxQuant analysis pipeline. Thus, TMTproC advances multiplexed proteomics data quality and widens access to accurate multiplexed proteomics beyond laboratories with MS3-capable instrumentation.
Insights
New TMTproC technology enhances multiplexed proteomics by improving quantification accuracy and sensitivity at the MS2 level. This method expands access to high-quality multiplexed proteomics without specialized instruments.
Area of Science:
- Proteomics
- Mass Spectrometry
- Biotechnology
Background:
- Multiplexed proteomics enables cell state analysis but faces challenges with accurate quantification due to co-isolated peptide interference.
- MS3-based quantification reduces interference but lowers sensitivity and requires specialized equipment.
- Complementary ion quantification offers accurate MS2-level multiplexing compatible with standard instruments but has limitations with TMT-tags.
Purpose of the Study:
- To evaluate and optimize complementary ion quantification using the TMTpro-tag (TMTproC) for enhanced multiplexed proteomics.
- To assess the performance of TMTproC in terms of plexing capacity, sensitivity, and accuracy compared to existing methods.
Main Methods:
- Utilized TMTpro-tag for complementary ion quantification, increasing multiplexing to eight channels (TMTproC).
- Evaluated TMTproC performance against TMTpro-MS3 and real-time-search SPS-MS3 (RTS-SPS-MS3) methods.
- Developed compatible software for TMTproC data analysis within the MaxQuant pipeline.
Main Results:
- TMTproC demonstrated increased sensitivity, quantifying approximately 65% more proteins than TMTpro-MS3 and 18% more than RTS-SPS-MS3.
- TMTproC exhibited superior accuracy compared to TMTpro-MS2 and RTS-SPS-MS3.
- The TMTproC method supports eight-channel multiplexing, overcoming limitations of previous TMT-tags.
Conclusions:
- TMTproC significantly advances multiplexed proteomics by enhancing data quality, accuracy, and sensitivity.
- This method broadens accessibility to precise multiplexed proteomics, particularly for labs without MS3-capable instrumentation.
- TMTproC represents a valuable tool for cell state analysis in health and disease research.
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