Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Sep;12(9):2623-39.
doi: 10.1074/mcp.M112.027078. Epub 2013 May 20.

Design, Implementation and Multisite Evaluation of a System Suitability Protocol for the Quantitative Assessment of Instrument Performance in Liquid Chromatography-Multiple Reaction monitoring-MS (LC-MRM-MS)

Affiliations
Free PMC article

Design, Implementation and Multisite Evaluation of a System Suitability Protocol for the Quantitative Assessment of Instrument Performance in Liquid Chromatography-Multiple Reaction monitoring-MS (LC-MRM-MS)

Susan E Abbatiello et al. Mol Cell Proteomics. .
Free PMC article

Abstract

Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation <0.15, peak width coefficient of variation <0.15, standard deviation of RT <0.15 min (9 s), and the RT drift <0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the use and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to ensure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread use of MRM-MS technology by the basic biomedical and clinical laboratory research communities.

Figures

Fig. 1.
Fig. 1.
Workflow for the development, evaluation and use of a system suitability protocol (SSP) for system performance. A, scheme of the method development process for picking peptides and transitions from a predefined sample, including generation of spectral libraries, generation of vendor-specific transition lists, data processing, and report generation in Skyline. Exported Skyline data reports were further analyzed to select the nine most appropriate peptides (based on criteria outlined in text) for use in the SSP for all sites. B, use of the SSP for evaluating system performance. The final nine peptides are acquired and the data analyzed in Skyline, from which the data metrics (peak area CV, normalized peak area CV, RT drift, RT standard deviation, and FWHM CV) can be directly exported in a report format to observe Pass/Fail status.
Fig. 2.
Fig. 2.
Evaluation of retention time stability from the SSP. A, RT drift observed for the 9 peptides on each of the 15 instrument platforms, grouped by LC manufacturer. Peptides are sorted in order of increasing retention time drift at each site. B, RT Viewer display of the injection-to-injection RT drift for peptide CAV (CAVVDVPFGGAK from glutamate dehydrogenase) at 14 selected sites. Sites 12, 14, and 15 show the largest drift in RT whereas sites 3, 8 and 10 show the smallest. Site 11 is not displayed as results were off scale due to problems with flow calibration. C, Skyline display of injection-to-injection variability in RT for one peptide at Site 10.
Fig. 3.
Fig. 3.
Comparison of normalized peak area in the system suitability sample across 15 sites. A, CV of normalized peak area versus the mean of the normalized peak area for each site for the nine final peptides (each box represents one peptide). B, injection-to-injection variability of normalized peak area. The Site Legend identifies sites by color and the type of MS instrument used at each site.
Fig. 4.
Fig. 4.
Skyline tools developed for rapid assessment of system suitability performance at a single site. Panel A: average peak area across 10 injections for each of the original 22 peptides monitored in the SSP (error bars represent 1 standard deviation). Peptides are sorted in order of retention time with retention time shown in the x axis. Panel B: CV of the peak areas for each peptide across 10 injections. Panel C: view of results for the raw peak area observed for peptide TAA for 10 replicate injections; each color representing the signal from the individual transitions.
Fig. 5.
Fig. 5.
Before and After Plots of SSP Data from Individual Sites with System Performance Problems. Panel A: data from two SSP peptides (CAVVDVPFGGAK and VGPLLACLLGR) that exhibited large retention time shifts (in blue), before room temperature stabilization at Site 10. When the sample was re-run, the “after” retention time shifts, shown in green, are more representative of the retention time variability observed at other sites. Panel B: unusually wide FWHM for peptide VLDALDSIK at Site 5, indicating a problem with the HPLC plumbing. After the connections were fixed, Site 5 obtained narrower FWHM more typical of the other sites. Panel C: before and after view of the Peak Area view in Skyline for Site 4. The last 5 peptides of the SS sample showed very low signal as compared with the other sites involved in the study. When Site 4 re-calibrated the flow meter on their organic solvent pump, the peak areas for the later eluting peptides increased and were observed to be more similar to the other sites. Panel D: same data from Site 4 as in Panel C but using the Peak Area CV view in Skyline. The later eluting peptides all have elevated CV values for Peak Area in the “before” case. When the system was fixed, all CVs dropped, but the effect was largest on the later eluting peptides.
Fig. 6.
Fig. 6.
Correlation of Peak Area CV of SS peptides and LOD of peptides from a quantitative SID-MRM-MS assay. Six sites acquired a 9-point response curve to determine LOD of 10 target peptides. The SSP was acquired at the beginning of the study and periodically throughout the response curve. Panel A: peak area CV of the SS peptides for all injections of the SS sample throughout the study for each site. Panel B: plot of the calculated LOD of 8 out of the 10 peptides from the quantitative study. The two peptides from CRP were not included as endogenous amounts were detected in the blank plasma matrix and LODs could not be accurately determined.

Similar articles

See all similar articles

Cited by 36 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback