Sample preparation strategies for targeted proteomics via proteotypic peptides in human blood using liquid chromatography tandem mass spectrometry.
Proteomics Clin Appl
; 9(1-2): 5-16, 2015 Feb.
Article
em En
| MEDLINE
| ID: mdl-25418444
ABSTRACT
The simultaneous quantification of protein concentrations via proteotypic peptides in human blood by liquid chromatography coupled to quadrupole MS/MS is an important field of bioanalytical research with a high potential for routine diagnostic applications. This review summarizes currently available sample preparation procedures and trends for absolute protein quantification in blood using LC-MS/MS. It discusses approaches of transferring established qualitative protocols to a quantitative analysis regarding their reliability and reproducibility. Techniques used to enhance method sensitivity such as the depletion of high-abundant proteins or the immunoaffinity enrichment of proteins and peptides are described. Furthermore, workflows for (i) protein denaturation, (ii) disulfide bridge reduction and (iii) thiol alkylation as well as (iv) enzymatic digestion for absolute protein quantification are presented. The main focus is on the tryptic digestion as a bottleneck of protein quantification via proteotypic peptides. Conclusively, requirements for a high-throughput application are discussed.
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MEDLINE
Assunto principal:
Fragmentos de Peptídeos
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Proteínas Sanguíneas
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Cromatografia Líquida
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Proteômica
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Espectrometria de Massas em Tandem
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Métodos Analíticos de Preparação de Amostras
Idioma:
En
Ano de publicação:
2015
Tipo de documento:
Article