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- W2267144736 abstract "Current methods and technologies to analyze complex proteomes rely on the use of costly instrumentation and separation tools on top of labor-intensive methods in order to more thoroughly characterize the representative proteins. Common methods to characterize proteomic specimens include geLC-MS, 2D-gel electrophoresis,MuDPIT, and off-gel electrophoresis (OGE) ahead of LC-MS/MS. We have evaluated an alternative method that does not require additional sample fractionation on additional equipment, meanwhile minimizing alterations introduced by analytical techniques. For both whole human cells and isolated mitochondria, the proposed method involved exposure of the pelleted cells or organelles to sequencegrade trypsin in order to obtain peptides and proteins cleaved from the cell and organelle surfaces. The “stripped” cells and mitochondria were also lysed and their contents digested. Additionally, total lysates of cells and mitochondria not exposed to trypsin ahead of lysis were generated and tryptically digested. The obtained proteomic profiling results were compared to those acquired using commonly employed fractionation techniques (OGE and SDS-PAGE). Resulting digests from all protocols were analyzed using LC-MS/MS, and all were analyzed for the proportion of unique and differentially enriched proteins achieved with each. Our results showed that the inexpensive and simplistic fractionation method allowed for similar or superior depth of proteomic profiling as well as a substantial increase in the number of unique identifications of membrane-bound and extracellular matrix related proteins. Additionally, the method provides information about spatial localization and differential abundance of proteome subsets in the cell or organelle architectural compartments. These results suggest that a very straightforward approach for the preparation of cellular and organellar specimens prior to proteomic characterization can be reproducibly and reliably utilized by biological and medical laboratories that have no previous analytical biochemistry expertise." @default.
- W2267144736 created "2016-06-24" @default.
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- W2267144736 date "2011-10-01" @default.
- W2267144736 modified "2023-09-28" @default.
- W2267144736 title "The Poor-Man's Fractionation of Cellular and Organellar Proteomes at the Stages of Lysis and Proteolytic Digestion, Eliminating the Use of Analytical Fractionation Techniques." @default.
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