Jay S. Johnson (Presenter)
Waters Corporation
Bio: Jay received his B.S. in Chemistry from The George Washington University and his M.S. in Analytical Chemistry from Northeastern University. In his current role as Senior Scientist in Waters' Core Research Group, he has supported the commercial release of two microfluidic solutions, TRIZAIC and ionKey/MS, acquiring extensive knowledge and expertise in microflow chromatography and the implementation of these approaches in a variety of applications including clinical research and health sciences.
Authorship: Jay S. Johnson(1), Morteza Razavi(2), James Murphy(1), Selena Larkin(2), Paul Rainville(1)
(1)Waters Corporation, Milford, MA. (2)SISCAPA Assay Technologies, Washington, DC.
| Short Abstract Use of an optimized SISCAPA enrichment that is highly specific for a signature peptide of thyroglobulin combined with microflow LC/MS using a vetted dual-pump trapping configuration provides a sub 1 ng/mL quantification limit of Tg protein with a cycle time of 6.75 min. This quantification limit is comparable with the best in literature for standard flow LC/MS. Microflow also offers other tangible benefits including the use of five times less starting plasma and half the injection volume of the standard flow method to reach this detection limit while being more precise. Accordingly, microflow is a viable and attractive solution for clinical research. |
Long Abstract
Thyroglobulin (Tg) is a 660 kDa dimeric protein proposed as a clinical research biomarker for evaluating treatment efficiency in thyroid cancer and recurrence. Current research immunoassays for Tg may be subject to high false negative rates in a significant portion of the sample population due to the presence of endogenous anti-Tg autoantibodies. The use of standard isotope standard and capture by anti-peptide antibodies (SISCAPA) enrichment of Tg tryptic peptides from human plasma, combined with the standard flow LC/MS has been implemented as an alternative approach in clinical research labs (1). SISCAPA avoids the potential for any deleterious reporting effects of auto antibodies due to a proteolytic digestion step. Furthermore the high analytical selectivity and specificity of the capture step using anti-peptide antibodies specific for a proteolytic peptide unique to Tg greatly enhances the detection and quantitiation of Tg down to levels of approximately 1 ng/mL (2). Microflow LC/MS, exemplified by the ionKey/MS system, operating at 10's of µL/min offers substantial analytical sensitivity benefits over standard flow using less starting plasma in sample-limited applications (3). Accordingly, we investigate here if the microflow system operating in a dual-pump trapping configuration can provide reductions in LLOQ levels for Tg using less plasma while maintaining the requisite accuracy, precision, and throughput exemplified by standard flow LC/MS assays.
Proteotypic peptides of Tg were derived from human plasma by digesting using trypsin. A unique peptide specific to Tg with the amino acid sequence, FSPDDSAGASALLR and the corresponding stable isotope standards (SIS) were selectively enriched from the digest via incubation with anti-peptide antibodies conjugated to magnetic beads. The beads were washed to remove unbound matrix material and then bound peptides released using acid elution. The resulting eluent was then subjected to microflow LC/MS analysis.
In all experiments, a dual-pump microflow LC/MS configuration was used to analyze the enriched samples at 3µL/min on a 150µm x 50mm microfluidic device integrated with an optimized ESI source. A pre-enrichment column was also utilized to allow for fast loading of 20µL of the extracts. Detection was performed using a triple quadrupole mass spectrometer operating in MRM mode. The dual-pump trapping configuration was explicitly chosen due to the ability of the set-up to handle relatively large injection volumes, reduce carryover, and decrease cycle time by affording load ahead capability on the trap column and independent washing and equilibration of the trap column and analytical column.
The analytical sensitivity of the microflow system was first evaluated using synthetic standards of the light and heavy FSP peptides. A 6 point calibration curve was created comprising a concentration range of 2,000 amol/µL down to 0.64 amol/µL. We observed an excellent linear response and reproducibility over the calibration range with the 13 amol on column level having a coefficient of variation of approximately 16%.
Next, to demonstrate that the platform is compatible with SISCAPA eluates and can actually detect endogenous Tg in human plasma we performed a plasma titration experiment followed by SISCAPA enrichment of the FSP peptide. The results shown the expected linear response was achieved for human plasma amounts down to 40µL. Furthermore, as a positive control, the experiment was replicated on an Agilent 1290/6490 QqQ instrument operating in the standard flow regime and utilizing the recommended method parameters for the instrument. A high linear correlation of R2 = 0.998 was achieved between the two platforms. Furthermore, the microflow system showed better precision across 4 replicates than the standard flow system. This suggests microflow offers tangible improvements in the precision of measurement of FSP while maintaining the accuracy expected of the conventional standard flow approach.
To further study the sensitivity of the platform in terms of LLOD and LLOQ, a reverse curve was generated by titrating the heavy FSP peptide from 5000 amol/µL down to 0.75 amol/µL and spiking synthetic light peptide at a constant level in human plasma. The LLOD, was determined to be 15 amol on column and the LLOQ was 45 amol.
A final curve was generated by titrating purified Tg protein in bovine plasma known to be deficient in Tg from 100 ng/mL to 0.1 ng/mL followed by SISCAPA enrichment in attempts to get an estimated LLOQ value for the entire assay including the digestion step. The LLOQ of the assay using only 50µL of plasma is estimated to be approximately 0.78 ng/mL.
Use of an optimized SISCAPA enrichment that is highly specific for a signature peptide of thyroglobulin combined with microflow LC/MS using a vetted dual-pump trapping configuration provides a sub 1 ng/mL quantification limit of Tg protein with a cycle time of 6.75 min. This quantification limit is comparable with the best in literature for standard flow LC/MS. Microflow also offers other tangible benefits including the use of five times less starting plasma and half the injection volume of the standard flow method to reach this detection limit. Additionally, evidence is provided in a head-to-head comparison with standard flow that the microflow approach offers highly correlated PAR measurements for Tg while being significantly more precise across 4 replicate measures. We therefore conclude that the configuration described does provide acceptable LLOQ levels for Tg using significantly less plasma while maintaining the requisite accuracy, better precision, and throughput levels exemplified by standard flow LC/MS assays. Accordingly, microflow is a viable and attractive solution for clinical research.
References & Acknowledgements:
(1)Kushnir, M.M., et al. Measurement of Thyroglobulin by Liquid Chromatography-Tandem Mass Spectrometry in Serum and Plasma in the Presence of Antithyroglobulin Autoantibodies. Clin. Chem. (2013) 59(6), 982.
(2)Pelttari H., Post-ablative Serun Thyroglobulin is an Independent Predictor of Recurrence in Low-risk Differentiated Thyroid Carcinoma: a 16-year Follow-up Study. Eur. J. Endocrinol. (2010) 163, 757.
(3)Lame, M.E., Chambers, E.E., Improving a High Sensitivity Assay for the Quantification of Teriparatide in Human Plasma Using the ionKey/MS System, Library Number APNT134788933, PN 720004948EN
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