(M1430-06-33) The Impact of Unique Stable Label Peptide Internal Standards toward Large Molecule Applications for LC-MS/MS

Purpose: Historically for LC-MS/MS based applications proteins or peptides undergo digestion cleaving a unique peptide used for downstream quantitation. Based on cost, generally unique peptide stable label internal standards are synthesized and used in these applications instead of full length stable label internal standards. Previously published work had suggested that unique peptide internal standards had minimal impact on accuracy of quantitation (Bronsema et al, 2012). This application was limited to the evaluation of a monoclonal antibody using direct digest. The conclusion of the report was that stable label internal standards may not be necessary for large molecule LC-MS/MS applications. The function of a unique peptide stable label internal standards within these applications is limited to tracking variability during digestion, although this is not always the case depending on the type of unique peptide internal standard selected. For peptide stable label internal standards containing no amino acid residues flanking the expected cleavage sites, the internal standard predominately corrects for variability in well to well ionization differences, Figure 1. We will discuss how unique peptide stable label internal standards function with respect to their impact on accuracy of quantitation across multiple modalities. Case studies assessing matrix effects, recovery and autosampler stability to demonstrate how use of unique peptide stable label internal standards can impact quantitation will be shown.

Methods: Matrices for each of the modalities was human K2-EDTA plasma. Protein biomarker: The unique peptide was extracted from the protein biomarker by means of enzymatic digestion. A sample of 25 uL was used for the assay. Waters Acquity BEH Shield RP18 operating at a gradient with post-elution wash-off with an initial flow rate of 0.350 mL/min was used for separation in conjunction with a triple quadrupole Sciex 5500. Modified protein: Extraction of the modality was carried out using immunoprecipitation with an anti-idiotypic capture antibody. 50µL of calibrators prepared in human EDTA plasma were pre-diluted. The KingFisher was used to elute the therapeutic agent. Samples enzymatically digested and quenched with acid. An Avantor ACE Excel 2 C18 column operating at a gradient with post-elution wash-off with an initial flow rate of 0.6 mL/min was used for separation in conjunction with a triple quadrupole Sciex API6500. Peptide: Extraction of the modality was carried out using protein precipitation. 150 uL of sample and 25 uL of peptide stable label internal standard was precipitated with 50:50 ACN:MeOH. The supernatant was evaporated to dryness under nitrogen, and reconstituted using 30:70 ACN:H2O. Waters Acquity Premier HSS T3 operating at a gradient with a flow rate of 0.700 mL/min was used for separation in conjunction with a triple quadrupole Sciex API6500.

Results: Stable label internal standards are assumed to be identical in structure when compared to the analyte, minimizing impact to quantitation if matrix effects or instability in sample extracts are encountered. However, if the internal standard is only a labeled representative portion of the intact compound, variability tracking and matrix effect correction may not occur. Instability in a sample extract can manifest as a reduction in response due to nonspecific binding, dimerization or an increased response if evaporation is occurring in the sample well. Table 1 shows results for stored extract stability across the three modalities at both low and high concentrations for the three respective assays. The peptide modality demonstrates no difference between analyte and peptide internal standard response; therefore, not impacting quantitation. The modified protein shows a substantial reduction in analyte and cleaved peptide internal standard response when compared to freshly prepare quality control samples. However, the area ratio was not impacted therefore the accuracy of quantitation was not impacted. The protein biomarker shows an inconsistent change in analyte compared to peptide stable label internal standard at the low concentration QC. In addition, apparent matrix effects were observed for the protein biomarker in disease state matrix when compared to healthy normal matrix.

Conclusion: The data demonstrate the need to critically evaluate data when incorporating the use of a unique peptide stable label regardless of modality. Recovery after digestion could be useful tool to assess if digestion successfully produces the same species in the final extract. Matrix effects could be assessed using standard addition if the modality is endogenous or recovery after digestion could also be used to confirm release of both the unique unlabeled and labeled peptide used for quantitation, thus providing confidence that there is no difference to between the analyte and internal standard in the extract.

References: K.J. Bronsema, et al., Journal of Chromatography B, 893-894 (2012), p. 1-14

Figure 1: Observed variability of a unique peptide stable label internal standard response over forty analytical runs.

Table 1. Response for stored samples extracts across the three modalities compared to the response of freshly extracted samples. The unique peptide stable label internal standards used for the peptide and modified protein show a reduction in response to the analytes. The unique peptide stable label internal standard used for the protein biomarker does not show a change in response proportional that of the analyte.