Background: Throughout the proteomics era, the analysis of serum as a source of biomarkers for disease risk and status has retained interest. For subsequent validations, targeted proteomics provides a powerful alternative to antibody based methods. Nevertheless, the throughput of the methodology can encumber its scope for application with large cohorts.
Data from analyses aimed towards increasing the throughput of targeted serum protein measurement are presented. Selected Reaction Monitoring (SRM) and Parallel-Reaction Monitoring (PRM) with a conventional capillary LC (75 µm) and an EvoSep system were used.
Methods: Serum was prepared for proteomics without depletion. Synthetic analogues for proteotypic peptides, including the PQ500 panel (Biognosys) and retention time standards, were used to evaluate separation and scheduling methods in terms of targets detected and analysis time. For library creation and PRM, a Q Exactive Orbitrap HF mass spectrometer was used. A TSQ triple quadrupole MS was used For SRM measurements. An EasyNano and an EvoSep LC instrument were evaluated.
Endogenous peptides and isotopically labelled synthetic peptides were measured throughout to confirm targets and control peak integration, and two stable unique peptides per protein were measured where possible.
Results: SRM of multiple targets was restricted by the number of concurrent transitions. However, evaluating several peptides per protein and maximizing use of the chromatographic time window reduced this limitation. PRM provided selective detection for a range of common protein targets with sensitivity comparable to SRM and excellent selectivity.
Comparing the LC systems, the EvoSep system enabled time saving for the moderate sized panels of targets, reducing loading and equilibration times.
Conclusions: With SRM and PRM measurements, in the order of 120 protein targets was achieved whilst maintaining two or more peptides per protein and recording multiple transitions or PRM spectra. In particular, the use of peptides across the chromatographic scale was an important advantage.