Purpose
Early diagnosis of primary immunodeficiency disorders (PIDD) is critical for maximizing patient survival and clinical outcomes. Consequently, there is significant interest in developing broad-based, high-throughput, screening approaches capable of utilizing small blood volumes to identify patients with PIDD.
Experimental Design
We developed a novel proteomic screening approach using tandem mass spectrometry to simultaneously identify specific signature peptides derived from the transmembrane protein CD3ε and the intracellular proteins WASP and BTK as markers of three life-threatening PIDDs; Severe Combined Immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS) and X-Linked Agammaglobulinemia (XLA). Signature peptides were analyzed by LC/MS-MS in proteolytically digested lysates from cell lines and white blood cells. The amount of each peptide was determined by the ratio of the signature peptide peak area to that of a known amount of labeled standard peptide. Peptide concentrations were normalized to Actin.
Results
We show that signature peptides from CD3ε, WASP, and BTK were readily detected in proteolytically digested cell lysate and their absence could correctly identify PIDD patients.
Conclusions and clinical relevance
This proof of concept study demonstrates the applicability of this approach to screen for PIDD and raises the possibility that it could be further multiplexed to identify additional PIDDs and potentially other disorders.
The redox status of mitochondrial coenzyme Q (CoQ) is an important marker for oxidative stress associated with several disorders such as Parkinson disease and Alzheimer disease. Altered redox status may be present in mitochondrial electron transport complex disorders. Intracellular CoQ levels reflect the functional status of the mitochondrial electron transport complex better than plasma levels. Here, we describe the method to determine the reduced and oxidized form of CoQ in white blood cells using LC-MS/MS.
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