Sunhee Jung (Presenter)
Seattle Children
Bio: Sunhee Jung is interested in biomedical application of mass spectrometry and quantitative analysis. Her current research focuses on developing MS-based tests to measure potential protein markers in the blood for genetic diseases such as primary immunodeficiency diseases, cystinosis and Wilson disease. She acquired a solid background in proteomics using mass spectrometry as well as molecular and cellular biology while earning her doctorate at the University of Washington.
Authorship: Sunhee Jung1, Jeffrey R. Whiteaker2, Lei Zhao2, Remwilyn Dayuha1, Troy Torgerson1,3, Amanda G. Paulovich2, and Si Houn Hahn1,3
1Seattle Children’s Hospital Research Institute, Seattle, WA; 2Fred Hutchison Cancer Research Center, Seattle, WA; 3Department of Pediatrics, University of Washington School of Medicine, Seattle, WA.
| Short Abstract Primary immunodeficiency disorders (PIDDs) are a diverse group associated with defective immune system. Because of the susceptibility to severe infections caused by the immunodeficiency, early detection of life-threatening PIDDs is critical for maximizing patient survival and clinical outcomes. Many of these treatable PIDDs are associated with the absence/reduced levels of a particular protein or a particular immune cell subset that can provide clues to a diagnosis. We herein report a proof-of-concept study demonstrating that the immuno-SRM can detect extremely low abundance marker proteins of CD3e, BTK, and WASP in DBS for three life-threatening PIDDs: Severe Combined Immudeficiency, X-linked Agammaglobulinemia, and Wiskott-Aldrich syndrome, respectively. Our promising data opens up the great potential of a multiplexed immuno-SRM assay for screening a variety of congenital disorders. |
Long Abstract
Introduction: Primary immunodeficiency disorders (PIDDs) are a diverse group associated with defective immune system. Early detection of life-threatening PIDDs is critical for maximizing patient survival and clinical outcomes because of the susceptibility to severe infections caused by the immunodeficiency. With the recent addition of Severe Combined Immunodeficiency (SCID) to the current NBS panel, the era of prospective screening for immune deficiencies began. The current approach to SCID screening utilizes quantitative real-time PCR to detect T cell Receptor Excision Circles (TREC). While effective, this DNA-based approach is applicable only to a subset of immunodeficiencies. Unfortunately, for most of the life-threatening but treatable, “non-SCID” immunodeficiencies, there are currently no broad-based, cost-effective screening methods available. Many of these treatable PIDDs are associated with the absence/reduced levels of a particular protein or a particular immune cell subset that can provide clues to a diagnosis. We hypothesized that peptide immunoaffinity enrichment coupled with Liquid Chromatography-Selected Reaction Monitoring-Mass Spectrometry (immuno-SRM) can be utilized as a rapid approach to detect various PIDDs using Dried Blood Spots (DBS). We performed a pilot study to evaluate this approach for the identification of three life-threatening PIDDs: SCID, Wiskott-Aldrich syndrome (WAS), and X-linked Agammaglobulinemia (XLA) and obtained preliminary data indicating that immuno-SRM can detect extremely low abundance marker proteins of CD3e (for SCID), BTK (for XLA), WASP (for WAS) in DBS. The results demonstrate proof-of-concept for applying immuno-SRM to screen for a variety of congenital disorders.
Methods: We developed rabbit polyclonal antibodies for two or three unique peptides for BTK, CD3e, and WASP, which we previously studied [1]. The polyclonal antibodies were used to enrich the corresponding peptides from tryptic digests of DBS. Peptides were analyzed in SRM mode with 6500 QTRAP (ABSCIEX). Fresh blood samples from two XLA patients were collected after consent was obtained and all research procedures were approved by the institutional review boards. Blood samples were spotted on filter paper, dried and stored in sealed plastic bags at -80 oC until use.
Preliminary Results: Using polyclonal antibodies, we analyzed target peptides for BTK, CD3e, and WASP in DBS from two normal controls and two XLA patients in a blinded fashion using multiplexed immuno-SRM. Preliminary results showed absence/markedly reduced levels of the two BTK target peptides in both XLA patients compared to normal controls, while the other target peptides designed to detect SCID and WAS (CD3e and WASP) in the XLA patients were at similar levels as the normal controls. Performance metrics of the assay were determined by generating a 6-point response curve using synthetic standard peptides. The assay showed a linear response (r2 ≥ 0.95) for five target peptides (two for BTK, one for CD3e, and two for WASP), spanning the peptide concentration of about 0.1 to 134 nmol/L. The median CV for all points on the response curve was 11%. Lower limits of quantitation, defined by the lowest point to yield CV<20%, ranged from about 50 to 93 pmol/L. The linear response with the high reproducibility shows constant protein recovery and protein digestion efficiency for the target peptides. Intra- and inter-assay imprecision is under evaluation by analyzing concentrations of endogenous peptides from a normal control DBS on 5 different days each using 5 replicates of the sample. Intra-assay imprecision for the 5 target peptides ranged from 11% to 22% CV. The stability of analytes in DBS at room temperature for various duration is currently under investigation. So far, our stability test showed the five target peptides are fairly stable in DBS up to 3 days at room temperature (percent differences ranged from 1 to 9% after 72 hours).
Conclusions: These data provide compelling evidence that immuno-SRM can be used as a highly sensitive platform for proteins that are present at low concentrations in DBS. As far as we know, this is the first quantitative analysis of these three low-abundance proteins in DBS by immuno-SRM. When multiplexed, there is a significant potential to screen various congenital disorders lacking specific protein markers such as various PIDDs, Cystinosis, and Wilson Disease. Further optimization efforts with monoclonal antibodies are underway to enable immuno-SRM as a viable platform for NBS. Our findings open up an innovative approach for NBS, facilitating the development of multiplexed screening tests for a broader range of genetic disorders.
References & Acknowledgements:
[1] Kerfoot, S. A.; Jung, S.; Golob, K.; Torgerson, T. R.; Hahn, S. H. Tryptic peptide screening for primary immunodeficiency disease by LC/MS-MS. Proteomics Clin. Appl. 2012, 6, 394.
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