期刊
JOURNAL OF PROTEOME RESEARCH
卷 17, 期 7, 页码 2480-2490出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.8b00235
关键词
sample preparation techniques; bottom-up proteomics; suspension trap; filter-aided sample preparation; sodium dodecyl sulfate; label-free quantification; digestion comparison; tandem mass spectrometry; quantitative proteomics
资金
- National Institutes of Health [R01GM110406]
- National Institutes of Health Training Grant-Chemistry Biochemistry Biology Interface Program [T32GM075762]
- National Science Foundation (CAREER Award) [CHE-1351595]
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM110406, T32GM075762] Funding Source: NIH RePORTER
Bottom-up proteomic strategies rely on efficient digestion of proteins into peptides for mass spectrometry analysis. In-solution and filter-based strategies are commonly used for proteomic analysis. In recent years, filter-aided sample preparation (FASP) has become the dominant filter-based method due to its ability to remove SDS prior to mass spectrometry analysis. However, the time-consuming nature of FASP protocols have led to the development of new filter-based strategies. Suspension traps (S-Traps) were recently reported as an alternative to FASP and in-solution strategies as they allow for high concentrations of SDS in a fraction of the time of a typical FASP protocol. In this study, we compare the yields from in-solution, FASP, and S-Trap based digestions of proteins extracted in SDS and urea-based lysis buffers. We performed label-free quantification to analyze the differences in the portions of the proteome identified using each method. Overall, our results show that each digestion method had a high degree of reproducibility within the method type. However, S-Traps outperformed FASP and in-solution digestions by providing the most efficient digestion with the greatest number of unique protein identifications. This is the first work to provide a direct quantitative comparison of two filter-based digestion methods and a traditional in solution approach to provide information regarding the most efficient proteomic preparation.
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