Analytical Considerations for the Development of Plate-Based Proteomics Platforms Using Isobaric Labeling.

Area of Science:

• Proteomics
• Biotechnology
• Analytical Chemistry

Background:

• Mass spectrometry-based proteomics platforms are crucial for biological research, drug discovery, and biomarker identification.
• Increasing demand for automated sample processing necessitates integrated automation platforms with liquid chromatography-mass spectrometers, forming high-throughput (HT) proteomics platforms.
• Quantitative proteome data at protein or peptide level is the output of these HT proteomics platforms.

Purpose of the Study:

• To develop a plate-based HT proteomics standard, termed HT-sKO, for evaluating platform accuracy and estimating quantification limits.
• To assess the impact of acquisition strategies and informant assays on HT proteomics platform stability and data quality.
• To demonstrate the performance and compatibility of the HT-sKO standard across different proteomic profiling methods.

Main Methods:

• Development of the HT-sKO standard using nonhuman recombinant proteins spiked into samples.
• Evaluation of platform accuracy and relative limit of quantification for proteins with up to 60-fold abundance variation.
• Assessment of intra- and inter-plate variance using the HT-sKO standard.
• Testing compatibility with whole-proteome, phospho-proteome, and reactive cysteine profiling.

Main Results:

• The HT-sKO standard allows accurate evaluation of quantification limits across a 60-fold protein abundance range.
• Intra- and inter-plate variance was found to be approximately 4-6% at the protein level and 10% at the peptide level.
• A robust acquisition strategy and tube-based informant assays were shown to be foundational for stable HT proteomics platforms.
• The HT-sKO standard demonstrated compatibility with diverse proteomic analysis types.

Conclusions:

• The developed HT-sKO standard provides a reliable method for assessing the performance of plate-based HT proteomics platforms.
• The standard facilitates improved accuracy and reproducibility in quantitative proteomic data acquisition.
• HT-sKO is a versatile tool compatible with various proteomic applications, supporting advancements in biological understanding and biomarker discovery.