Quantification of ATP7B Protein in Dried Blood Spots by Peptide Immuno-SRM as a Potential Screen for Wilson’s Disease

Jung, Sunhee; Whiteaker, Jeffrey R.; Zhao, Lei; Yoo, Han‐Wook; Paulovich, Amanda G.; Hahn, Si Houn

doi:10.1021/acs.jproteome.6b00828

Cited by 47 publications

(31 citation statements)

References 71 publications

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“…To improve the sensitivity and reproducibility of the assay, we harnessed the immuno-SRM platform to quantify very low abundance peptides in DBS such as surrogate peptides of the ATP7B protein from patients with Wilson disease (WD). Our results demonstrated immuno-SRM's capability to detect ATP7B peptides in the low picomolar (pmol) range and reproducibly differentiating between patients with WD from unaffected controls (33). The goal of our present study is to expand this immuno-SRM method to the multiplexed analysis of a large cohort of patients with SCID, WAS, or XLA.…”

Section: Introductionmentioning

confidence: 71%

Rapid Multiplexed Proteomic Screening for Primary Immunodeficiency Disorders From Dried Blood Spots

Collins¹,

Chang

Jung³

et al. 2018

Front. Immunol.

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Background: Primary immunodeficiency disorders (PIDD) comprise a group of life-threatening congenital diseases characterized by absent or impaired immune responses. Despite the fact that effective, curative treatments are available with optimal clinical outcomes when diagnosed early, newborn screening does not exist for the majority of these diseases due to the lack of detectable, specific biomarkers or validated methods for population-based screening. Peptide immunoaffinity enrichment coupled with selected reaction monitoring mass spectrometry (immuno-SRM) is a sensitive proteomic assay, involving antibody-mediated peptide capture, that allows for concurrent quantification of multiple analytes. This assay has promise for use in potential newborn screening of PIDDs that lead to diminished or absent target proteins in the majority of cases.Objective: To determine and evaluate if a multiplex assay based on immuno-SRM is able to reliably and precisely distinguish affected patients with X-linked agammaglobulinemia (XLA), Wiskott-Aldrich Syndrome (WAS), and CD3ϵ-associated severe combined immunodeficiency (SCID) from one another and from unaffected normal control dried blood spot (DBS) samples.Methods: We performed a blinded, multiplexed analysis of proteolytically-generated peptides from WASp, BTK, and CD3ϵ (for WAS, XLA, and SCID, respectively) in DBS samples from 42 PIDD patients, 40 normal adult controls, and 62 normal newborns. The peptide ATPase copper transporting protein (ATP7B) 1056 was simultaneously monitored for quality assurance purposes.Results: The immuno-SRM assays reliably quantified the target peptides in DBS and accurately distinguished affected patients from normal controls. Analysis of signature peptides found statistically significant reduction or absence of peptide levels in affected patients compared to control groups in each case (WASp and BTK: p = 0.0001, SCID: p = 0.05). Intra and inter-assay precision ranged from 11 to 22% and 11 to 43% respectively; linearity (1.39–2000 fmol peptide), and stability (≤ 0.09% difference in 72 h) showed high precision for the multiplexed assay. Inter-laboratory assay comparison showed high concordance for measured peptide concentrations, with R2 linearity ≥ 0.97 for the WASp 274, CD3ϵ 197, BTK 407, and ATP7B 1056 peptides.Conclusion: Immuno-SRM-based quantification of proteotypic peptides from WASp, BTK, and CD3ϵ in DBS distinguishes relevant PIDD cases from one another and from controls, raising the possibility of employing this approach for large-scale multiplexed newborn screening of selective PIDDs.

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Section: Introductionmentioning

confidence: 71%

Rapid Multiplexed Proteomic Screening for Primary Immunodeficiency Disorders From Dried Blood Spots

Collins¹,

Chang

Jung³

et al. 2018

Front. Immunol.

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“…However, recent efforts do suggest the applicability towards larger biomolecules177,178 . Some investigations has also utilized DBS and DMS for proteomic investigations as concept studies87,[179][180][181][182] . Nevertheless, DMS and DBS are not extensively reported for analysis of low abundance biomarkers.…”

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confidence: 99%

Instant on-paper protein digestion during blood spot sampling

Skjærvø

Rosting

Halvorsen

et al. 2017

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A concept integrating sampling and protein digestion is introduced here combining fast and simple fabrication by wax printing on filter paper with trypsin immobilized polymer beads. The paper reactors showed promising results with a high degree of protein digestion within fifty minutes in model protein mixtures as well as in human blood. The model protein mixture was used for the evaluation of performance both with and without a reduction and alkylation step. The paper reactors without reduction and alkylation showed between 46% and 75% protein sequence coverage and between five and 20 high confidence peptides (one and five zero missed cleavage peptides, respectively). Compared to a conventional in-solution approach, the paper reactor showed 10% less protein sequence coverage, 29% fewer high confidence peptides and 19% fewer high confidence peptides with zero missed cleavages. Placement of the protein reduction and alkylation step (before or after protein digestion) was shown to be of low importance. The storage stability of the paper reactors with (six weeks) and without (twelve weeks) tryptic peptides was satisfactory. The ability of the paper reactors to digest complex biological samples was investigated by comparison with human whole blood samples prepared using a conventional dried blood spot (DBS) procedure with overnight digestion in non-targeted analysis. The reactors showed a comparable performance with 75 ± 25 for the protein groups compared to 76 ± 5 for the DBS samples. Additionally, 267 ± 72 and 335 ± 11 unique peptides (high confidence) were identified for on-paper digestion and DBS, respectively.

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“…Each patient in the blinded study was deficient in the signature peptide specific for their respective disease [i.e., XLA patient lacking Bruton's Tyrosine Kinase (BTK) and WAS patient missing WAS protein (WASP), etc.]. These efforts were subsequently extended to include peptide immunoaffinity enrichment coupled to selected reaction monitoring (Immuno-SRM) technology (15,27), also referred to as Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA). Immuno-affinity enrichment of signature peptide biomarkers using anti-peptide antibodies isolates peptides of interest from complex biological matrices.…”

Section: Introductionmentioning

confidence: 99%

Multiplexed Proteomic Analysis for Diagnosis and Screening of Five Primary Immunodeficiency Disorders From Dried Blood Spots

Collins¹,

Dayuha²,

Whiteaker

et al. 2020

Front. Immunol.

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Early detection of Primary Immunodeficiencies Disorders (PIDDs) is of paramount importance for effective treatment and disease management. Many PIDDs would be strong candidates for newborn screening (NBS) if robust screening methods could identify patients from dried blood spots (DBS) during the neonatal period. As majority of congenital PIDDs result in the reduction or absence of specific proteins, direct quantification of these target proteins represents an attractive potential screening tool. Unfortunately, detection is often limited by the extremely low protein concentrations in blood cells and limited blood volume present in DBS. We have recently developed a robust novel method for quantification of low abundance proteins in DBS for PIDDs using peptide immunoaffinity enrichment coupled to selected reaction monitoring (immuno-SRM). Here, we further generated a multiplexed Immuno-SRM panel for simultaneous screening of eight signature peptides representing five PIDD-specific and two cell-type specific proteins from DBS. In samples from 28 PIDD patients including two carriers, representing X-Linked Agammaglobulinemia (XLA), Wiskott-Aldrich Syndrome (WAS), X-Linked Chronic Granulomatous Disease (XL-CGD), DOCK8 Deficiency and ADA deficiency, peptides representing each disease are significantly reduced relative to normal controls and patient identification had excellent agreement with clinical and molecular diagnosis. Also included in the multiplex panel are cell specific markers for platelets (CD42) and Natural Killer Cells (CD56). In patients with WAS, CD42 levels were found to be significantly reduced consistent with characteristic thrombocytopenia. A patient with WAS analyzed before and after bone marrow transplant showed normalized WAS protein and platelet CD42 after treatment highlighting the ability of immuno-SRM to monitor the effects of PIDD treatment. The assay was readily reproduced in two separate laboratories with similar analytical performance and complete agreement in patient diagnosis demonstrating the effective standardized methods. A high-throughput Immuno-SRM method screens PIDD-specific peptides in a 2.5-min runtime meeting high volume NBS workflow requirements was also demonstrated in this report. This high-throughput method returned identical results to the standard Collins et al. Proteomic Screening for Primary ImmunodeficienciesImmuno-SRM PIDD panel. Immuno-SRM peptide analysis represents a robust potential clinical diagnostic for identifying and studying PIDD patients from easily collected and shipped DBS and supports a significant potential for early PIDD diagnosis through newborn screening.

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Quantification of ATP7B Protein in Dried Blood Spots by Peptide Immuno-SRM as a Potential Screen for Wilson’s Disease

Cited by 47 publications

References 71 publications

Rapid Multiplexed Proteomic Screening for Primary Immunodeficiency Disorders From Dried Blood Spots

Rapid Multiplexed Proteomic Screening for Primary Immunodeficiency Disorders From Dried Blood Spots

Instant on-paper protein digestion during blood spot sampling

Multiplexed Proteomic Analysis for Diagnosis and Screening of Five Primary Immunodeficiency Disorders From Dried Blood Spots

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