Protein precipitation of diluted samples in SDS-containing buffer with acetone leads to higher protein recovery and reproducibility in comparison with TCA/acetone approach.
Proteomic approaches are extremely valuable in many fields of research, where mass spectrometry methods have gained an increasing interest, especially because of the ability to perform quantitative analysis. Nonetheless, sample preparation prior to mass spectrometry analysis is of the utmost importance. In this work, two protein precipitation approaches, widely used for cleaning and concentrating protein samples, were tested and compared in very diluted samples solubilized in a strong buffer (containing SDS). The amount of protein recovered after acetone and TCA/acetone precipitation was assessed, as well as the protein identification and relative quantification by SWATH-MS yields were compared with the results from the same sample without precipitation. From this study, it was possible to conclude that in the case of diluted samples in denaturing buffers, the use of cold acetone as precipitation protocol is more favourable than the use of TCA/acetone in terms of reproducibility in protein recovery and number of identified and quantified proteins. Furthermore, the reproducibility in relative quantification of the proteins is even higher in samples precipitated with acetone compared with the original sample.
Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.|Institute for Interdisciplinary Research, University of Coimbra, Coimbra, Portugal.
Cátia
C
Santa
Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.|Faculty of Sciences and Technology, University of Coimbra, Coimbra, Portugal.
Sandra I
SI
Anjo
Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.
Bruno
B
Manadas
D000096:Acetone / Q000737:chemistry*; D002021:Buffers; D011232:Chemical Precipitation; D002853:Chromatography, Liquid / Q000379:methods; D011489:Protein Denaturation; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods; D014238:Trichloroacetic Acid / Q000737:chemistry*
D002021:Buffers; D011506:Proteins; D000096:Acetone; D012967:Sodium Dodecyl Sulfate; D014238:Trichloroacetic Acid
Acetone; Protein precipitation; SWATH; TCA/acetone; Technology