title: Sample preparation strategies for targeted proteomics via proteotypic peptides in human blood using liquid chromatography tandem mass spectrometry.
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.
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany; LIFE - Leipzig Research Center for Civilization Diseases, University Leipzig, Leipzig, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Dittrich
authors.forename: Susen
authors.initials: S
authors.lastname: Becker
authors.forename: Max
authors.initials: M
authors.lastname: Hecht
authors.forename: Uta
authors.initials: U
authors.lastname: Ceglarek
mesh_terms: D053000:Analytic Sample Preparation Methods; D000818:Animals; D001798:Blood Proteins / Q000032:analysis*; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D010446:Peptide Fragments / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
chemical_list: D001798:Blood Proteins; D010446:Peptide Fragments; D014357:Trypsin
keywords: Absolute quantification of proteins; Proteotypic peptides; Quantitative proteomics; Sample pretreatment; Tryptic digestion