Comparison of SPEED, S-Trap, and In-Solution based sample preparation methods for tandem mass spectrometry

ABSTRACT: Bottom-up proteomic analyses rely on efficient protein extraction from tissue and proteolysis into peptides for mass spectrometry. Commonly used detergent-based strategies aid cell lysis and protein solubilization but are poorly compatible with downstream protein digestion and liquid chromatography-coupled mass spectrometry. Consequently, additional sample purification and buffer exchange steps are required, which are time-consuming and introduce additional technical variability. This study provides the first direct quantitative comparison of two well-established detergent-based methods for protein extraction and solubilization (In-solution and suspension trapping, S-Trap) with the recently developed Sample Preparation by Easy Extraction and Digestion (SPEED) method, which uses a strong acid for denaturation. Identification rates and quantitative performance of each method were compared with data-dependent acquisition (DDA) and label-free SWATH-MS (sequential window acquisition of all theoretical mass spectra) in both sheep kidney cortical tissue and plasma. In kidney tissue, SPEED outperformed In-solution and S-Trap by quantifying the highest number of unique proteins (SPEED 1,250; S-Trap 1,202; In-solution 1,197) and displayed the most efficient proteolysis (SPEED 93.8% of peptides fully tryptic in DDA; S-Trap 88.5%; In-solution 87.3%). In plasma, S-Trap produced the most unique protein quantifications (S-Trap 151; In-solution 148; SPEED 138), indicating it may be the optimal method for this biofluid. Protein quantifications were reproducible across biological replicates in both tissue (R2 from 0.85 to 0.90), and in plasma (SPEED R2=0.84; In-solution R2=0.76, S-Trap R2=0.65). Our data suggest SPEED as the optimal method for proteomic preparation in kidney tissue and S-Trap or SPEED as the optimal method for plasma, depending on whether a higher number of protein quantifications or greater reproducibility is desired.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Ovis Aries

TISSUE(S): Blood Plasma, Kidney

SUBMITTER: Evie Templeton  

LAB HEAD: Victoria Ann Cameron

PROVIDER: PXD030797 | Pride | 2023-03-30

REPOSITORIES: Pride