Evaluation of prioritized peptide acquisition for multiplexed single-cell proteomics on an Orbitrap Astral Zoom MS

Significance of the topic

The development of sensitive, high-throughput single-cell proteomics workflows addresses a critical gap in cellular biology and clinical research: measuring proteome-level heterogeneity at true single-cell resolution. Multiplexed isobaric labeling combined with prioritized acquisition strategies can increase throughput and data completeness while preserving depth of coverage, enabling comparative studies of rare cell populations such as leukemic blasts and leukemia stem cells (LSCs). This evaluation demonstrates practical performance limits and optimization routes for routine, large-scale single-cell proteomics.

Objectives and study overview

The primary aim was to evaluate a prioritized peptide acquisition (pSCoPE/pDDA) workflow for multiplexed single-cell proteomics implemented on a Thermo Scientific Orbitrap Astral Zoom mass spectrometer. Goals included assessing throughput (cells/day), proteome depth per cell, sensitivity for small leukemic cells (AML8227 model), comparison with conventional DDA, and practical considerations for target list generation and labeling strategies using TMTPro 35plex.

Methodology

Key elements of the experimental workflow:

• Single-cell sample handling: AML8227 single cells (blast and LSC subpopulations) were FACS-sorted into 1 µL of 20% TFE, 80 mM TEAB, 0.01% DDM in 384-well plates and frozen. Digestion was performed in 1 µL buffer containing 2 ng/µL trypsin at 37 °C overnight.
• Isobaric labeling and pooling: Digests were labeled with TMTPro reagents (1 µL at 1.9 µg/µL in acetonitrile), quenched with hydroxylamine, pooled, acidified, dried, and resuspended in 2% ACN/0.1% TFA. A bulk dilution of AML8227 cells provided reference channels.
• Multiplex layout: Typical sample sets contained 31 single-cell channels (e.g., 16 blasts, 15 LSC) plus two reference channels (127N and 127D) to normalize between deuterated and non‑deuterated labels.
• Prioritized acquisition strategy: Priority lists were generated from a prior DIA analysis and organized into three tiers per FAIMS compensation voltage (CV). Lists were ordered by precursor abundance and could be filtered by biological relevance, peptide count per protein, and best-performing peptides. Per-peptide parameters included retention-time windows and an intensity threshold (10% in this study).
• LC and MS method: Analyses used a 30 seconds-per‑day (30 SPD) rapid method on a Vanquish Neo UHPLC with a 50 cm μPAC Neo Plus column operated in trap-and-elute mode. A dedicated ddMS2 branch was created for each priority list and FAIMS CV.

Used instrumentation

• Mass spectrometer: Thermo Scientific Orbitrap Astral Zoom Mass Spectrometer.
• UHPLC: Thermo Scientific Vanquish Neo UHPLC System.
• Column: 50 cm Thermo Scientific μPAC Neo Plus HPLC Column in trap-and-elute configuration.
• Labels: TMTPro 35plex reagents (including deuterated variants).
• Software: Thermo Scientific Proteome Discoverer 3.4 with CHIMERYS algorithm (default settings).
• Reference FASTA: Human Swiss-Prot canonical sequences (cited 20,528 entries for HeLa runs; human proteome reference used for identification).

Data analysis

All raw data were processed in Proteome Discoverer 3.4 using the CHIMERYS search algorithm with default settings. Priority lists originated from DIA runs and were tailored to each FAIMS CV. Identification and quantification relied on TMTPro reporter ions. Retention-time windows and intensity thresholds were applied to focus acquisition on predefined peptide targets and improve triggering efficiency.

Results and discussion

Key findings from evaluation of the prioritized acquisition workflow:

• Throughput: Using the 30 SPD method, extrapolated throughput exceeds 1,000 single cells per day, demonstrating high practical capacity for large studies.
• Proteome depth: On average, 1,452 proteins were quantified per AML8227 single cell without employing a carrier channel, indicating strong sensitivity even for relatively small leukemic cells.
• Identification success and comparison to DDA: Prioritized SCoPE (pSCoPE) delivered a substantially higher identification success rate (48%) versus conventional DDA (14%), and quantified 26% more proteins than DDA despite DDA acquiring ~3× more spectra. This indicates the efficiency of targeted triggering for low-input samples.
• PSM and TMT performance: Reporter signal abundances varied across channels and between cells—reflecting biological heterogeneity such as cell size and cell-cycle stage—yet mean and median reporter intensities were high, supporting robust TMT-based quantification at single-cell scale.
• Practical optimizations: The study highlighted the importance of refining target peptide lists and tightening retention-time windows to increase the number of successfully triggered peptide MS2 events, thereby improving peptide and protein quantification counts.

Benefits and practical applications

The evaluated workflow offers several practical advantages for laboratories pursuing single-cell proteomics:

• High throughput suitable for population-scale single-cell studies and comparative analyses between subpopulations (e.g., blasts vs. LSCs).
• Enhanced identification efficiency and proteome coverage via prioritized acquisition, enabling quantification without carrier channels and reducing potential carrier-related artifacts.
• Compatibility with 35‑plex TMTPro labeling allows dense multiplexing and flexible experimental designs, including use of deuterated labels to differentiate cell types.

Future trends and potential applications

Opportunities to further improve and apply the workflow include:

• Refinement of priority lists: Automated selection of optimal peptides (best-responding proteotypic peptides) and dynamic RT-window calibration will increase triggered targets and depth.
• Scaling and experimental design: Replacing dual reference channels by labeling strategies that separate cell types by deuteration could free channels and allow analysis of up to 35 single cells per TMT set, further increasing per-run throughput.
• Integration with advanced search algorithms and real‑time acquisition control to adapt targets during runs, improving identifications for rare or low-abundance proteins.
• Adoption in clinical and translational workflows for profiling rare cell populations, patient stratification, and monitoring cellular responses to therapies at proteome level.

Conclusion

The prioritized peptide acquisition workflow implemented on the Orbitrap Astral Zoom MS combined with TMTPro 35plex labeling and optimized LC conditions provides a practical route to high-throughput, high-sensitivity single-cell proteomics. The approach quantifies over a thousand cells per day in projection, yields ~1,450 proteins per AML8227 cell on average without a carrier, and outperforms conventional DDA in identification success and quantified protein counts. Continued optimization of target lists and retention-time precision will further increase triggered events and overall proteome coverage.

References

1. Budnik et al., 2018 – original SCoPE-MS publication (isobaric single-cell proteomics approach cited).
2. Huffman et al., 2023 – prioritized targeting (pSCoPE) methodology referenced.
3. Proteome Discoverer 3.4 and CHIMERYS (Thermo Fisher Scientific) – software used for data processing.