Evaluating First-Pass, High Protein Capacity Desalting Techniques For Phosphoproteomics Applications.

Area of Science:

• Proteomics
• Biochemistry
• Analytical Chemistry

Background:

• Commercial desalting kits are available for peptide cleanup before mass spectrometry (MS).
• Few kits effectively handle high protein input (≥ 4 mg) for phosphotyrosine enrichment.
• Technical expertise is often a barrier for new users of desalting products.

Purpose of the Study:

• To evaluate four commercial desalting techniques for protein profiling and phosphotyrosine (pY) enrichment.
• To determine the most cost-effective, user-friendly, and sensitive method for high-throughput proteomics.
• To assess methods based on automation, organization, and chemistry.

Main Methods:

• Comparison of four commercial desalting techniques: TECAN NBE, ProtiFi S-Trap, Pierce C18 spin columns, and one unspecified method.
• Evaluation of cost-effectiveness, user-friendliness, and sensitivity for peptide sequencing and pY enrichment.
• Application in a Jurkat T cell signaling model using Src SH2 superbinder enrichment.

Main Results:

• ProtiFi S-Trap demonstrated superior performance in peptide sequencing and protein profiling, identifying 25,654 unique peptides and 375 unique proteins.
• TECAN NBE was most cost-effective per sample and easiest to use.
• Pierce C18 spin columns exhibited the poorest operational organization.

Conclusions:

• ProtiFi S-Trap columns offer high peptide recovery, significantly enhancing the identification of biologically relevant phosphotyrosine sites.
• The choice of desalting method impacts the depth and breadth of proteomic and phosphoproteomic analyses.
• ProtiFi S-Trap is recommended for sensitive phosphotyrosine enrichment and comprehensive protein profiling.