Plasma preparation to measure FDA‐approved protein markers by selected reaction monitoring

Abstract

Background The development of commercially available panels for human blood plasma screening via selected reaction monitoring (SRM) offers reliable, cost-efficient and highly-standardized discovery and validation of protein biomarkers. However, protein detection by SRM can be hampered by interfering peptide fragment ions. To estimate the influence of interference on protein detection, we performed different types of sample preparation and implemented SRM measurements for well-characterized protein targets approved by the US Food and Drug Administration. Methods We used the PlasmaDeepDive™ SRM assay from BiognoSYS AG for absolute quantification of 18 proteins in 19 samples of human plasma using three different protocols for sample preparation. SRM measurements were performed using iRT standards for retention time normalization and isotopically-labeled reference peptides for absolute quantification. SpectroDive™ software was used for automated detection of reliable peak groups. Results Fourteen targeted proteins were quantitatively measured in more than half of the samples. Depletion of highly-abundant plasma proteins and peptide fraction clean-up on centrifuge plates resulted in detection of all 18 targeted proteins in femtomolar to picomolar concentrations. Conclusions It was shown that commercially designed SRM kits are suitable for SRM detection of well-established plasma/serum biomarkers. Electronic supplementary material The online version of this article (doi:10.1186/s40169-015-0071-4) contains supplementary material, which is available to authorized users.

Food allergen detection by mass spectrometry: the role of systems biology

“…One potential avenue for achieving greater sensitivity is the immunoaffinity depletion of high abundance protein, as is common for SRM plasma biomarker quantitation. 62 , 63 This could prove to be a useful avenue of further research, especially for foods characterized by a dominant protein-contributing ingredient; for example, depleting wheat protein in breads or cookies when quantitating milk proteins.…”

Systematic evaluation of immune regulation and modulation

“…The protein digestion can be performed using trypsin at 37 °C for three hours with stirring. The trypsinization is then terminated by adding trichloroacetic acid, the pH adjusted, the trypsinized plasma is dried, resolved in liquid chromatography solution, spiked with isotypically labeled peptide standard and then used for mass spectrometric analysis [ 28 ]. Sample collection and preparation are critical steps to obtain useful information in clinical proteomics analyses.…”

Plasma prefractionation methods for proteomic analysis and perspectives in clinical applications

“…Indeed, utilizing the undepleted plasma, in the same way as immunological based assays, would be the ideal solution to bypass all of the above issues. However, studies showed that MRM‐MS of the undepleted plasma typically had the lower LOD and the lower LOQ in the high nanogram per milliliter to low microgram per milliliter range of target proteins , while the LOD and LOQ could be extended to low nanogram per milliliter range by applying plasma prefractionation prior to MS analysis . It seems that in MS‐based assay plasma prefractionation should be considered and performed depending on the concentration range of target proteins and the sensitivity of MS platforms.…”