TMT MS3 and SPS-MS3: How to Avoid Ratio Compression to Enhance Quantitative Accuracy?
In TMT multiplexed proteomics experiments, researchers often encounter a core issue: ratio compression. Due to the co-elution of homologous peptides from different samples at the MS1 level and the presence of co-isolation background ion interference, the intensity of TMT reporter ions is often diluted, leading to an underestimation of the true quantitative differences. To address this issue, mass spectrometer manufacturers and methodological experts have proposed MS3 and SPS-MS3 techniques, which significantly improve TMT quantification accuracy on the Orbitrap platform. This article will analyze the causes of ratio compression, the principles of MS3/SPS-MS3, the differences between them, and their applicable scenarios.
1. Causes and Impact of Ratio Compression
1. Definition of Ratio Compression
In TMT quantitative experiments, if the true abundance difference of the same peptide between two sample groups is 10-fold, the final reporter ion signal may only show 5-6 fold. This underestimated difference is referred to as ratio compression.
2. Main Causes
(1) Co-isolation interference: Non-target peptides within the MS/MS fragmentation window (usually 0.7–1.2 Th) contribute additional reporter ion signals;
(2) Non-specific fragmentation products: Background peptides release reporter ions during CID/HCD processes, causing quantification to be diluted.
3. Impact on Data
(1) Differential proteins are underestimated or cannot be identified, increasing false negative rates;
(2) It introduces bias to downstream biological interpretations, such as pathway enrichment analysis.
2. Principles of TMT MS3 Technology
1. Limitations of MS2 Quantification
Quantification directly using reporter ion intensity at the MS2 level is easily affected by co-elution interference, leading to ratio compression.
2. MS3 Solution
(1) Select fragment ions of the target peptide at the MS2 level;
(2) Re-excite these fragments at the MS3 level (usually through HCD) to generate second-generation fragments;
(3) Reporter ion signals originate only from the target peptide, improving quantification specificity.
3. Effect and Cost
(1) Significantly reduces ratio compression, improving quantification accuracy;
(2) Scanning speed decreases, and peptide coverage is somewhat reduced, especially in complex samples.
3. Optimization of SPS-MS3 Technology
1. SPS (Synchronous Precursor Selection) Mechanism
(1) At the MS3 level, multiple (usually 6–10) MS2 fragment ions are selected as precursors;
(2) These ions are synchronously excited to generate combined reporter ion signals.
2. Advantages
(1) Enhanced reporter ion signal intensity (higher signal-to-noise ratio);
(2) Mitigates the issue of insufficient signal from a single peptide, balancing sensitivity and accuracy;
(3) Significantly reduces ratio compression, especially in highly complex samples.
3. Applicable Scenarios
(1) Large cohort projects requiring high-precision differential quantification;
(2) Analysis of low-abundance proteins or complex background samples such as exosomes and serum;
(3) Publication-level projects with high accuracy and reliability requirements.
4. Comparison Summary of MS3 and SPS-MS3
1. Quantification Accuracy
SPS-MS3 > MS3 > MS2。
2. Data Coverage
MS3/SPS-MS3 has longer scanning times and slightly lower peptide coverage, but the SPS strategy partially alleviates the decrease in sensitivity.
3. Instrument and Method Requirements
(1) Requires high-end Orbitrap platforms (Fusion Lumos, Exploris series);
(2) SPS-MS3 demands higher method optimization and instrument tuning.
5. How to Choose the Appropriate Strategy?
1. If the core need of the project is differential expression accuracy (e.g., drug target validation, clinical biomarker screening), SPS-MS3 is recommended.
2. If focusing on coverage or if there are many samples with limited budget, MS2 combined with strict co-isolation interference filtering strategies can be used.
3. For exosomes, low-abundance samples, or large cross-batch projects, SPS-MS3 can help avoid false negatives due to signal dilution to some extent.
6. BaiTai Bio's SPS-MS3 Solution
BaiTai Bio offers an SPS-MS3 TMT workflow based on the Orbitrap Fusion Lumos platform to meet researchers' high-precision needs, featuring:
① Significantly reduced ratio compression, enhancing quantification accuracy;
② Compatibility with 18plex high-throughput TMT projects, balancing sensitivity and coverage;
③ Strict quality control and batch standardization processes to ensure result consistency;
④ Providing a one-stop service from sample processing, mass spectrometry detection, to multi-dimensional data analysis (differential analysis, pathway enrichment, network construction).
Ratio compression is one of the main challenges in TMT quantitative proteomics, and the application of MS3 and SPS-MS3 technologies offers an effective solution for high-precision quantification. By combining advanced mass spectrometry platforms with optimized workflows, researchers can obtain more realistic and reliable differential expression data while ensuring sensitivity. For more information on the SPS-MS3 TMT workflow or to plan high-precision, multi-channel proteomics research, please contact BaiTai Bio for detailed solutions and professional support.
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