TMT Workflow on the Q Exactive Series -Instrument Parameter Optimization and Data Analysis in Proteome Discoverer 2.1 Software

Significance of the topic

Isobaric mass tagging with Tandem Mass Tags (TMT) has become a cornerstone technique in quantitative proteomics, enabling simultaneous relative quantification of multiple samples with high throughput and minimized experimental variance. Achieving accurate and sensitive quantification across six or ten channels demands carefully optimized instrument parameters and data analysis workflows. The Thermo Scientific Q Exactive series, when coupled with Proteome Discoverer 2.1 software, provides the platform to realize robust TMT workflows for complex biological samples.

Objectives and study overview

This application note aimed to establish an optimized acquisition and processing method for TMTsixplex and TMT10plex experiments on Q Exactive Plus and Q Exactive HF mass spectrometers. Key goals included refining sample preparation, liquid chromatography gradients, mass spectrometry settings, and Proteome Discoverer data processing to maximize peptide and protein identification rates while ensuring high quantification accuracy across dynamic sample mixtures.

Methodology and instrumentation

A standard E. coli digest and a HeLa protein digest were labeled with TMTsixplex or TMT10plex reagents. Samples were combined in defined ratios, desalted, and analyzed by nanoLC using an EASY-nLC 1000 system with PepMap C18 trapping and EASY-Spray analytical columns. Data were acquired in data-dependent mode on:

• Q Exactive Plus (70,000 MS1 resolution; 30,000 MS2 for TMTsixplex, 35,000 for TMT10plex)
• Q Exactive HF (120,000 MS1 resolution; 30,000 MS2 for TMTsixplex, 60,000 for TMT10plex)

The SequestHT and consensus workflows in Proteome Discoverer 2.1 incorporated static TMT modifications, co-isolation thresholds, S/N-based reporter quantification, and isotopic impurity corrections. Study-specific quantification methods and FASTA databases were configured, and consensus factors (e.g., channel, isolation width) were defined for ratio calculation.

Main results and discussion

Optimizing chromatographic gradients from 2 to 4 hours improved peptide separation and identifications, yielding up to 17% more peptides. Matching MS2 maximum injection time (100 ms) to resolving power transients maximized parallelization, boosting low-abundance quantification. Increasing MS2 AGC to 1e5 and using NCE 32 enhanced reporter ion S/N without over-fragmentation. Narrow precursor isolation windows (0.7 Th) reduced co-isolation, improving accuracy in the presence of background interference.

Benefits and practical applications of the method

The optimized workflow delivers high proteome coverage and precise quantification for up to ten conditions in a single run. It supports biological studies requiring multiplexed comparison, such as time-course experiments, drug response profiling, and biomarker discovery.

Future trends and applications

Advances in synchronous precursor selection (SPS) MS3 on Orbitrap Tribrid instruments promise further improvements in dynamic range and interference removal. Integration of high-field Orbitrap technology, enhanced pre-fractionation strategies, and streamlined software pipelines will continue to expand the applicability of TMT-based proteomics to increasingly complex biological systems.

Conclusion

This study provides a step-by-step protocol for TMTsixplex and TMT10plex experiments on Q Exactive Plus and HF platforms. By fine-tuning LC gradients, MS2 parameters, and Proteome Discoverer workflows, users can achieve maximal identification rates and reliable quantification across diverse sample types.

Instrumentation used

• Thermo Scientific Q Exactive Plus mass spectrometer
• Thermo Scientific Q Exactive HF mass spectrometer
• Thermo Scientific EASY-nLC 1000 system
• Thermo Scientific PepMap C18 and EASY-Spray columns

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