Combining Data Independent Acquisition With Spike-In SILAC (DIA-SiS) Improves Proteome Coverage and Quantification

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Data-independent acquisition (DIA) offers advantages over data-dependent acquisition (DDA) in proteomics, including higher throughput and reduced missing values.
• While DIA typically uses label-free quantification, integrating stable isotope labeling can enhance accuracy.
• Existing labeling methods like chemical tags or in-vivo SILAC have limitations in sample preparation or applicability.

Purpose of the Study:

• To develop and validate a novel method combining DIA with Spike-in Stable isotope labeling by amino acids in cell culture (DIA-SiS).
• To assess the performance of DIA-SiS for proteome profiling, particularly in challenging samples.
• To improve proteome coverage and quantification accuracy compared to existing label-free DIA approaches.

Main Methods:

• Development of the DIA-SiS workflow, integrating DIA acquisition with a Spike-in SILAC strategy.
• Performance evaluation using mixed-species benchmark samples at both bulk and single-cell levels.
• Application of DIA-SiS to analyze low-input formalin-fixed paraffin-embedded (FFPE) tissue sections.

Main Results:

• DIA-SiS significantly enhanced proteome coverage and quantification accuracy compared to label-free DIA.
• The method reduced the number of incorrectly quantified proteins.
• DIA-SiS demonstrated effectiveness in analyzing low-input FFPE tissue samples, showcasing its broad applicability.

Conclusions:

• DIA-SiS provides a robust and accurate method for comprehensive proteome profiling.
• This approach combines the benefits of stable isotope-based quantification with simplified, label-free sample preparation.
• DIA-SiS facilitates accurate and efficient proteomic analysis across various sample types, including challenging clinical specimens.