A High-Sensitivity Low-Nanoflow LC-MS Configuration for High-Throughput Sample-Limited Proteomics

Area of Science:

• Proteomics
• Mass Spectrometry
• Biochemistry

Background:

• High-throughput proteomics is crucial for analyzing limited biological samples.
• Optimizing nanoLC-MS workflows is essential for balancing sensitivity and speed.

Purpose of the Study:

• To demonstrate the utility of high-throughput nanoLC-MS and label-free quantification (LFQ) for sample-limited bottom-up proteomics.
• To optimize conditions for single-cell proteomics (SCP) analysis.
• To evaluate different data acquisition strategies for proteome coverage.

Main Methods:

• Optimization of a 50 μm I.D. column nanoLC-MS system at 100 nL/min.
• Evaluation of data-dependent acquisition (DDA), wide-window acquisition (WWA), and wide-window data-independent acquisition (WW-DIA).
• Implementation of a trap-and-elute workflow for reduced cycle times.

Main Results:

• Achieved >3,000 protein group identifications with WW-DIA using a 10-min gradient (72 samples/day).
• Reduced method cycle time to 14.4 min (100 samples/day) via trap-and-elute workflow.
• Identified >1,700 protein groups in library-free DIA analysis of single-cell samples.

Conclusions:

• The study provides a high-sensitivity, high-throughput nanoLC-MS configuration for sample-limited proteomics.
• The optimized method is suitable for demanding applications like single-cell proteomics.
• This approach significantly enhances proteome coverage and sample throughput.