期刊
JOURNAL OF PROTEOME RESEARCH
卷 7, 期 9, 页码 3860-3867出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr800161x
关键词
ultrasound; O-18 labeling; bottom up proteomics; tryptic digestion
资金
- NIH National Center for Research Resources [RR018522]
- NIH National Cancer Institute [R21 CA12619-01]
- Pacific Northwest National Laboratory's (PNNL) Laboratory Directed Research and Development Program
- Battelle [DE-AC05-76RLO1830]
A new sample processing workflow that uses high intensity focused ultrasound to rapidly reduce and alkylate cysteines, digest proteins and then label peptides with O-18 was developed for quantitative proteomics applications. Each step was individually refined to minimize reaction times, peptide loses and undesired byproducts or modifications. When this novel workflow was used, mouse plasma proteins were successfully denatured, alkylated, in-solution digested, and O-18-labeled in <10 min for subsequent analysis by liquid chromatography-electrospray ionization high resolution mass spectrometry. Performance was evaluated in terms of the number of mouse plasma peptides and proteins identified in a shotgun approach and the quantitative dynamic range. The results were compared with previously published results obtained using conventional sample preparation methods and were found to be similar. Advantages of the new method include greatly simplified and accelerated sample processing, as well as being readily amenable to automation.
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