Strategies for acquisition and processing of N-term acetylated peptides on the timsTOF Ultra 2

Significance of the Topic

Protein N-terminal acetylation is one of the most widespread post-translational modifications, shaping protein stability, localization and interaction networks. In plant systems, the complexity of plastid proteins and extensive proteolytic processing pose analytical challenges. Enhanced methods to profile N-terminal acetylation are critical to understand regulatory roles in photosynthesis, stress response and development.

Objectives and Study Overview

This study evaluated acquisition and data processing strategies to deepen coverage of N-terminal acetylated peptides in Arabidopsis thaliana extracts using the timsTOF Ultra 2 mass spectrometer. Two precursor selection schemes (default polygon and an adapted polygon including singly charged precursors above m/z 700) were compared. Sample preparation modes (SCX fractionated pool versus crude extract) and match-between-runs (MBR) processing were also assessed. A dedicated pipeline, Nta-Quant, was implemented to quantify peptide groups sharing the same N-terminus.

Methodology and Instrumentation

Proteome Preparation and Enrichment:

• Arabidopsis thaliana shoot extracts were split into two aliquots: a crude tryptic digest and an SCX-fractionated pool labeled with acetic anhydride-d6.
• SCX fractionation yielded ten fractions, which were pooled for analysis.

Liquid Chromatography and Mass Spectrometry:

• nanoElute 2 system with Aurora Ultimate CSI 25 cm column operated at 50 °C and 250 nL/min over a 45-minute gradient.
• timsTOF Ultra 2 mass spectrometer in PASEF DDA mode employing default and adapted precursor selection polygons.

Data Processing:

• MSFragger and IonQuant within FragPipe 22 were used for peptide identification and quantification.
• Match-between-runs (MBR) was applied to recover additional peptide identifications.
• The Nta-Quant Python pipeline aggregated peptides into N-terminal groups and quantified validated sites.

Main Results and Discussion

Precursor Selection Impact:

• Adapted polygon captured singly charged peptides between 5 and 11 amino acids, increasing unique peptide groups by approximately 8%.
• Nearly 13% of validated N-terminal acetylation sites were identified exclusively with the adapted acquisition scheme, highlighting its value for short peptides.

Sample Preparation Comparison:

• SCX-fractionated samples provided deep N-terminome coverage, identifying the majority of acetylation sites.
• Analysis of crude extracts, though less deep, contributed an additional ~4% of unique N-terminal sites, demonstrating complementary information.

Effect of MBR:

• Implementing match-between-runs increased the total quantified peptide groups by ~8%, reinforcing its utility in comprehensive profiling.

Practical Benefits and Applications

The combined strategy of tailored precursor selection, SCX enrichment and MBR processing delivers a more complete view of the N-terminome. This approach enables detection of low-abundance, short N-terminal peptides that are otherwise overlooked, facilitating studies of proteolytic processing, protein maturation and acetyltransferase specificity in plant biology and beyond.

Future Trends and Opportunities

Advances may include dynamic polygon adjustment using real-time feedback, integration of complementary fragmentation techniques (e.g. ETD), and expansion to other organisms or cell types. Automated workflows and machine learning–driven data analysis could further enhance throughput and depth, supporting large-scale N-terminome mapping in complex biological systems.

Conclusion

This work demonstrates that combining an adapted precursor selection strategy, SCX-based enrichment and match-between-runs processing on the timsTOF Ultra 2 significantly improves N-terminal acetylation profiling. The approach captures unique short peptides and enhances site coverage, providing a robust framework for detailed N-terminome studies.

Reference

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