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

Abstract

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.

Multiplexed measurement of protein biomarkers in high-frequency longitudinal dried blood spot (DBS) samples: characterization of inflammatory responses

“…We therefore selected two additional proteins whose concentrations are remarkably stable in number of proteins not used in the normalization showed CVs below 2.5%, close to the limit achievable with current MRM mass spectrometry for peptides. These CVs are generally much lower than most published work with DBS, in which CVs for individual protein assays typically range from 7-20% [41,44,45,47,51]. We attribute this improvement in precision mainly to the combination of highly specific immuno-affinity MS (which effectively separates the peptides analytes from digest matrix) and volume normalization (which has not been available in previous studies where appropriate normalizing proteins were not measured).…”

“…near-absolute structural specificity; facile, non-interfering multiplex measurement of many peptides; and direct analyte detection with true internal standards (a form of classical isotope dilution mass spectrometry frequently used to establish clinical reference methods). A number of studies have applied MRM methods [43][44][45][46][47][48] to DBS, demonstrating a capability to measure numerous proteins with reasonable precision and limited throughput. When directed to specific well-characterized peptides, a method combining MS detection with specific immuno-affinity peptide enrichment (SISCAPA; [5,8,32]) significantly enhances the sensitivity, throughput and linear dynamic range of the approach and delivers clinical-quality results [14], making it possible to measure broad panels of proteins in small samples with high precision and at low cost.…”

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

“…An operationally simple mass spectrometry assay using a less invasively collected sample by heel stick would allow for rapid screening of suspected PIDDs. The sensitivity and specificity of tandem mass spectrometry (MS/MS) based proteomic assay can be utilized to reliably measure extremely low abundance peptides in DBS extracts and thus quantify the proteins they represent (15,16). Furthermore, an assay capable of detecting PIDD patients using DBS would be applicable in newborn screening (NBS) and allow for patient identification before the onset of potentially fatal infections.…”

“…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.…”

“…This technique is highly reproducible, multiplexable, and transferrable across laboratories (34)(35)(36)(37)(38). Using this methodology in a blinded screen of 82 samples (42 patient samples with 40 normal controls), every molecularly confirmed case of XLA (n = 26), WAS (n = 11) and 2 out of 3 cases of SCID were significantly reduced in their respective peptides and diagnostic cutoffs allowed for their positive identification (15).…”