Interlaboratory reproducibility of selective reaction monitoring assays using multiple upfront analyte enrichment strategies

Abstract

Over the past few years, mass spectrometry has emerged as a technology to complement and potentially replace standard immunoassays in routine clinical core laboratories. Application of mass spectrometry to protein and peptide measurement can provide advantages including high sensitivity, the ability to multiplex analytes, and high specificity at the amino acid sequence level. In our previous study, we demonstrated excellent reproducibility of mass spectrometry-selective reaction monitoring (MS-SRM) assays when applying standardized standard operating procedures (SOPs) to measure synthetic peptides in a complex sample, as lack of reproducibility has been a frequent criticism leveled at the use of mass spectrometers in the clinical laboratory compared to immunoassays. Furthermore, an important caveat of SRM-based assays for proteins is that many low-abundance analytes require some type of enrichment before detection with MS. This adds a level of complexity to the procedure and the potential for irreproducibility increases, especially across different laboratories with different operators. The purpose of this study was to test the interlaboratory reproducibility of SRM assays with various upfront enrichment strategies and different types of clinical samples (representing real-world body fluids commonly encountered in routine clinical laboratories). Three different, previously published enrichment strategies for low-abundance analytes and a no-enrichment strategy for high-abundance analytes were tested across four different laboratories using different liquid chromatography-SRM (LC-SRM) platforms and previously developed SOPs. The results demonstrated that these assays were indeed reproducible with coefficients of variation of less than 30% for the measurement of important clinical proteins across all four laboratories in real world samples.

Figures

Figure 1

Protocol used in the study. Four different types of sample preparation (three enrichment protocols and one nonenrichment protocol) were used.

Figure 2

Breakdown curve plotted for six transitions of peptide SVILLGR. The response of a transition at a particular collision energy value is obtained by calculating the area under the peak of the SRM at that CE.

Figure 3

Scheduled SRM assay for 10 peptides from PSA. (A) The start and stop times for the various peptides that were monitored, and (B) the number of transitions that were being acquired at any given time during the acquisition.

Figure 4

Amounts of DKSVILLGR (representing PSA) calculated by the four laboratories in samples N, C, and P.

Figure 5

Amounts of the two isoforms of PSA calculated by the four laboratories in the MSIA-enriched clinical sample. SVILLGR and DTIVANP uniquely measure isoform 3 and isoform 4, respectively.

Figure 6

SRM peak AUC values of peptide 46S2+ from pituitary hGH spiked into urine with and without nanoparticle enrichment. A concentration curve was obtained by serial dilution into a normal urine sample and subjected to nanoparticle capture, or untreated. A magnified view of the lower concentration range is shown in the inset graph.

Figure 7

Amounts of FPTIPLSR (peptide representing hGH) calculated by the four laboratories in the nanoparticle-enriched clinical urine sample.

Figure 8

Amounts of four peptides (corresponding to four plasma proteins LDHC, PTGDS, SPAG11B, and FAM12B) calculated in the four laboratories in one seminal plasma sample.