Comparison of SPEED, S-Trap, and In-Solution-Based Sample Preparation Methods for Mass Spectrometry in Kidney Tissue and Plasma

Abstract

Mass spectrometry is a powerful technique for investigating renal pathologies and identifying biomarkers, and efficient protein extraction from kidney tissue is essential for bottom-up proteomic analyses. Detergent-based strategies aid cell lysis and protein solubilization but are poorly compatible with downstream protein digestion and liquid chromatography-coupled mass spectrometry, requiring additional purification and buffer-exchange steps. This study compares two well-established detergent-based methods for protein extraction (in-solution sodium deoxycholate (SDC); suspension trapping (S-Trap)) with the recently developed sample preparation by easy extraction and digestion (SPEED) method, which uses strong acid for denaturation. We compared the quantitative performance of each method using label-free mass spectrometry in both sheep kidney cortical tissue and plasma. In kidney tissue, SPEED quantified the most unique proteins (SPEED 1250; S-Trap 1202; SDC 1197). In plasma, S-Trap produced the most unique protein quantifications (S-Trap 150; SDC 148; SPEED 137). Protein quantifications were reproducible across biological replicates in both tissue (R2 = 0.85–0.90) and plasma (SPEED R2 = 0.84; SDC 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.

Non-mechanical disruption ofBacillus subtilis sporesallows sensitive and deep characterization of the minimal proteome for resuming a cellular lifestyle

“…Thus, complete lysis and protein extraction require additional attention, when designing a proteomics workflow for spores. Although, the novel approach to sample preparation without mechanical lysis SPEED has been applied to various of biological materials amongst which are bacterial vegetative cells (31,45,46), its performance in bacterial spores has not yet been assessed.…”

“…Thus, complete lysis and protein extraction require additional attention, when designing a proteomics workflow for spores. Although, the novel approach to sample preparation without mechanical lysis SPEED has been applied to various of biological materials amongst which are bacterial vegetative cells (31, 45, 46), its performance in bacterial spores has not yet been assessed. Comparative analysis of the protein profiles from B. subtilis cells and spores using SPEED sample preparation versus methods we established previously for bacterial spores (SP3 and OP) were first performed.…”

Development of a rapid and reliable method to simultaneously detect seven food allergens in processed foods using LC-MS/MS

“…However, LC-MS/MS analyses generally require 4–24 h for protease digestion of proteins extracted from food samples, and as processed foods contain many non-protein matrix components, such as lipids, salts, pigments, and polysaccharides, solid-phase extraction is often required to pre-purify and concentrate the target peptides, a process that complicates the analysis. In shotgun proteomic analyses, in which proteins in blood or cells are the analytical targets, several simple spin-column–type pre-treatment kits have been developed ( Templeton et al, 2023 ). These kits enable protein digestion and peptide purification using only centrifugation.…”