Optimization of digestion parameters for protein quantification.
Anal Biochem
; 393(1): 48-55, 2009 Oct 01.
Article
em En
| MEDLINE
| ID: mdl-19501563
ABSTRACT
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 2.51), 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.
Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Ricina
/
Bioquímica
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Glicoproteínas de Hemaglutininação de Vírus da Influenza
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Virus da Influenza A Subtipo H5N1
Tipo de estudo:
Evaluation_studies
Idioma:
En
Ano de publicação:
2009
Tipo de documento:
Article