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- Betancourt, Lazaro, et al.
(författare)
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Quantitative Assessment of Urea In-Solution Lys-C/Trypsin Digestions Reveals Superior Performance at Room Temperature over Traditional Proteolysis at 37 °C.
- 2018
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 17:7, s. 2556-2561
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Tidskriftsartikel (refereegranskat)abstract
- Urea-containing buffer solutions are generally used in proteomic studies to aid protein denaturation and solubilization during cell and tissue lysis. It is well-known, however, that urea can lead to carbamylation of peptides and proteins and, subsequently, incomplete digestion of proteins. By the use of cells and tissues that had been lysed with urea, different solution digestion strategies were quantitatively assessed. In comparison with traditional proteolysis at 37 °C, urea in-solution digestion performed at room temperature improved peptide and protein identification and quantitation and had a minimum impact on miscleavage rates. Furthermore, the signal intensities and the number of carbamylated and pyroglutamic acid-modified peptides decreased. Overall, this led to a reduction in the negative effects often observed for such modifications. Data are available via ProteomeXchange with identifier PXD009426.
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| 2. |
- Murillo, Jimmy Rodriguez, et al.
(författare)
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Mass spectrometry evaluation of a neuroblastoma SH-SY5Y cell culture protocol
- 2018
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697. ; 559, s. 51-54
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Tidskriftsartikel (refereegranskat)abstract
- Cell line-based proteomics studies are susceptible to intrinsic biological variation that contributes to increasing false positive claims; most of the methods that follow these changes offer a limited understanding of the biological system. We applied a quantitative proteomic strategy (iTRAQ) to detect intrinsic protein variation across SH-SY5Y cell culture replicates. More than 95% of the quantified proteins presented a coefficient of variation (CV) < 20% between biological replicates and the variable proteins, which included cytoskeleton, cytoplasmic and housekeeping proteins, are widely reported in proteomic studies. We recommend this approach as an additional quality control before starting any proteomic experiment.
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