کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1175073 961786 2009 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Optimization of digestion parameters for protein quantification
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Optimization of digestion parameters for protein quantification
چکیده انگلیسی

We present a rapid and efficient in-solution enzymatic digestion protocol suitable for mass spectrometry-based absolute protein quantification techniques. The digestion method employs RapiGest SF (an acid-labile surfactant), an excess amount of modified trypsin (enzyme-to-substrate ratio of 2.5:1), and an incubation time of 2 h. No reduction/alkylation reagents are used. Digestion parameters were varied systematically to monitor their effect on rate and completeness of digestion. To demonstrate the general applicability of the method, the optimization was done using a viral hemagglutinin (HA) as a model protein and then applied to ricin, a potent protein toxin extracted from the castor bean (Ricinus communis). The parameters that were optimized included incubation time, concentration of RapiGest SF, enzyme-to-substrate ratio, and incubation temperature. The optimization was done by comparing the yields from two protein-specific peptides originating from two different sites of the HA protein. The analysis was performed by liquid chromatography–tandem mass spectrometry in multiple reaction monitoring mode using isotopically labeled peptide standards for quantification.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 393, Issue 1, 1 October 2009, Pages 48–55
نویسندگان
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