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- Title
Comparison of SPEED, S-Trap, and In-Solution-Based Sample Preparation Methods for Mass Spectrometry in Kidney Tissue and Plasma.
- Authors
Templeton, Evelyn M.; Pilbrow, Anna P.; Kleffmann, Torsten; Pickering, John W.; Rademaker, Miriam T.; Scott, Nicola J. A.; Ellmers, Leigh J.; Charles, Christopher J.; Endre, Zoltan H.; Richards, A. Mark; Cameron, Vicky A.; Lassé, Moritz
- 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.
- Subjects
MASS spectrometry; INDUCTIVELY coupled plasma mass spectrometry; PROTEOLYSIS; KIDNEYS; SPEED; SAMPLING methods; AFFINITY chromatography; DENATURATION of proteins
- Publication
International Journal of Molecular Sciences, 2023, Vol 24, Issue 7, p6290
- ISSN
1661-6596
- Publication type
Article
- DOI
10.3390/ijms24076290