Russell P. Grant, Patricia M. Holland, Christopher M. Shuford
Laboratory Corporation of America
To better understand the inter-relationship of calibration (matrix and standard) and internal standardization on the accuracy of digestion-based protein quantification, the relative digestion efficiency of multiple internal standard types (peptides/protein), multiple standardized forms of unlabeled protein (recombinant/human-derived) and multiple matrices (true/surrogate) are evaluated for quantifying thyroglobulin in serum using multiple signature peptides. Comparing multiple matrices and protein standards will differentiate if the disparities/similarities between peptides are matrix effects and/or specific to the analytical standard used. A variety of digestion conditions are explored in combination with different internal standards to determine if disparities between peptides can be mitigated.
A PC-IDMS assay was developed and validated to CAP/CLIA guidelines for quantification of thyroglobulin in serum using signature peptide FSPDDASAVLLR (“FSP”) in combination with a cleavable SIL peptide analogue as the internal standard. The assay was externally calibrated using Beckman Access® Thyroglobulin Standard prepared in a proxy matrix consisting of 60 mg/mL HSA in PBS. The resulting assay was shown to provide good correlation with the FDA-approved Beckman Access® immunoassay in TgAb-negative patients (Deming Slope = 0.9462, R = 0.9784, N = 154).
In a subset of samples (N = 47), thyroglobulin concentration assignments were also performed using a second signature peptide, VIFDANAVPR (“VIF”), also using a cleavable SIL analogue as its internal standard. The VIF and FSP peptides were co-purified from digested calibrators and serum samples. Interestingly, the VIF peptide provided systematically higher results compared to the FSP peptide in serum samples (mean bias = 22%, p < 0.00001). In this cohort of specimens, the FSP again demonstrated good correlation with the Beckman Access® immunoassay (Deming Slope = 1.0456, R = 0.9757), while the VIF peptide was positively biased relative to the immunoassay (Deming Slope = 1.2191, R = 0.9817).
To confirm the discrepancy was not due to insufficient digestion time for peptide formation, a time course study was performed in multiple serum specimens as well as calibrators. In all cases, a plateau was observed in the formation of both the signature and SIL peptides before the 30 minute time period used in the assay digestion procedure. These data suggest that the discrepancy in the protein concentration assignments by the two peptides is due to peptide-specific digestion efficiencies that are different in the calibration and sample systems, which are not being normalized/corrected by the cleavable internal standards being deployed.
To examine the effect of internal standardization further, protein concentration assignments for thyroglobulin in 44 serum samples by both peptides were subsequently compared using three forms of internal standard 1) SIL peptides added post-digestion, 2) cleavable SIL peptides added pre-digestion (as above), and 3) recombinant full-length SIL thyroglobulin (SIL-Tg, Sigma-Aldrich®) added pre-denaturation. Each internal standard was uniquely labeled such that all could be implemented in parallel while being differentiated from one another based on their unique SRM transitions.
Utilizing SIL peptides added post-digestion, the VIF peptide produced systematically higher results than the FSP peptide in serum samples. The VIF peptide produced protein concentration assignments 31% higher on average than the FSP peptide (p < 0.00001), despite good correlation between these results (R = 0.9969) – indicating this combination of calibration system and internal standards were not correcting for disparities in the digestion process for the two peptides between calibrators and samples. Similarly, in using the cleavable SIL peptides added pre-digestion, concentration assignments from the VIF and FSP peptide showed good correlation (R = 0.9953), while the VIF peptide produced value 23% higher on average than the FPS peptide (p < 0.0001) – consistent with 22% mean bias observed in the previous study above. When comparing results obtained with the SIL-Tg added pre-denaturation, the VIF and FSP peptides showed improved agreement with the VIF peptide producing protein concentration assignments only 14% higher on average than the FSP peptide (p = 0.0045), while maintaining good correlation (R = 0.9864). Nonetheless, these results suggest a systematic bias between peptides produced in the calibration and sample systems, which is not wholly corrected even by the SIL-Tg internal standard.
A comparison of the light:heavy response ratios measured in samples using the SIL-Tg revealed almost no bias between the two peptides (mean bias = -3%, p = 0.115). Indeed, identical light:heavy ratios should be observed for all peptides produced from the light and heavy protein forms given the molar equivalency is ensured for all peptides within a given protein (PTMs notwithstanding). This is provided that 1) all light:heavy peptide pairs are detected using identical SRM transitions (as was done here) and 2) that the light and heavy proteins digest with the same efficiency. Thus, the lack of systematic bias in the light:heavy response ratios between the two peptides in serum samples suggests that the SIL-Tg digests with the same efficiency as the native serum Tg.
In contrast, the light:heavy response ratios measured in calibrators, and used to generate calibration curves, were different between the two peptides. This is exemplified by disparate slopes in the respective calibration equations for each peptide (linear, 1/x weighted): VIF-slope = 0.305, FSP-slope = 0.359. The 15% slope-bias in the calibration equations is consistent with the magnitude and direction of the bias observed for the Tg concentration assignments, indicating that the unlabeled Tg present in calibrators does not digest with the same efficacy as the SIL-Tg and, thus, that the unlabeled Tg present in calibrators does not digest with the same efficacy native Tg in serum.
To corroborate this finding, additional studies are underway to evaluate the relative digestion efficiency of the SIL-Tg and multiple standardized Tg materials in a variety of matrices. Evaluation of multiple matrices and Tg standards will differentiate if the findings are a matrix effect specific to the use of a proxy matrix for manufacturing of calibrators and/or specific to the protein (form) used as the analytical standard. Moreover, a variety of digestion conditions will be explored to determine if the observations are unique to the denaturation/digestion conditions employed.