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Sample preparation strategies for targeted proteomics via proteotypic peptides in human blood using liquid chromatography tandem mass spectrometry
Author(s) -
Dittrich Julia,
Becker Susen,
Hecht Max,
Ceglarek Uta
Publication year - 2015
Publication title -
proteomics – clinical applications
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.948
H-Index - 54
eISSN - 1862-8354
pISSN - 1862-8346
DOI - 10.1002/prca.201400121
Subject(s) - chemistry , chromatography , bottom up proteomics , quantitative proteomics , sample preparation , tandem mass spectrometry , bioanalysis , proteomics , mass spectrometry , liquid chromatography–mass spectrometry , protein mass spectrometry , biochemistry , gene
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.