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.

Targeting breast cancer-associated fibroblasts to improve anti-cancer therapy

“…However, many studies have been performed when proteomics was still in its exploratory phase. The introduction of standard sample preparation procedures [12] and quality control methods such as false discovery rate calculation [13] in addition to establishing the first mass spectrometry-based draft of the human proteome [14] are just about to consolidate the use of mass spectrometry for proteome analyses. We suggest that proteomics is now entering the application phase capable of coping with clinical challenges.…”

Clinical mass spectrometry proteomics (cMSP) for medical laboratory: What does the future hold?

“…This topic was recently reviewed, and the authors reached the conclusion that it is difficult to give general recommendations and that all the steps involved need to be carefully checked for each protein of interest [27]. Of note, when protocols involved manipulating very small volumes (<5μL) one should not forget that the random error can be as high as 10%, and that the repetition of these steps may contribute significantly to the inaccuracy of the method by propagating random errors.…”

Quality control in mass spectrometry‐based proteomics

“…Due to the wide variety in sample origin and experimental applications, each with their specific peculiarities and points of attention, it is impractical to cover all existing sample preparation techniques. However, appropriate sample preparation steps for a bottom‐up LC‐MS experiment typically include denaturation with a chaotrope, reductant, and/or alkylating agent followed by tryptic digestion of the proteins before the resulting peptides can be further processed . All these different steps will introduce a certain degree of variability in the output results, which needs to be monitored and controlled.…”

“…However, appropriate sample preparation steps for a bottom-up LC-MS experiment typically include denaturation with a chaotrope, reductant, and/or alkylating agent followed by tryptic digestion of the proteins before the resulting peptides can be further processed. 54,55 All these different steps will introduce a certain degree of variability in the output results, which needs to be monitored and controlled. As there are often multiple ways to conduct a single step to optimize the sample preparation a careful performance evaluation of the alternative options is required.…”