Journal
PROTEOMICS CLINICAL APPLICATIONS
Volume 9, Issue 1-2, Pages 5-16Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/prca.201400121
Keywords
Absolute quantification of proteins; Proteotypic peptides; Quantitative proteomics; Sample pretreatment; Tryptic digestion
Funding
- LIFE - Leipzig Research Center for Civilization Diseases, Universitat Leipzig
- European Union
- European Regional Development Fund (ERDF)
- Free State of Saxony within the framework of the excellence initiative
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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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