MSACL 2016 US Abstract

Differential MS and Targeted MS/MS Used to Identify an Aberrant ACTH Isoform

Mari DeMarco (Presenter)
Univ. of British Columbia, St Paul's Hospital

Bio: Dr. Mari DeMarco completed her PhD in the Biomolecular Structure and Design program at the University of Washington in Seattle, and a clinical chemistry fellowship at Washington University School of Medicine. She is a Clinical Chemist at St Paul's Hospital, and a Clinical Assistant Professor in the Department of Pathology and Laboratory Medicine, as well as the Associate Program Director for the Medical Biochemistry Residency Training Program at the University of British Columbia in Vancouver Canada.

Authorship: Pawan Dhaliwal (1), Yu Zi (Emma) Zheng (1), Mari L. DeMarco (1,2)
(1) University of British Columbia, Department of Pathology and Laboratory Medicine, Vancouver, Canada (2) St Paul’s Hospital, Department of Pathology and Laboratory Medicine, Vancouver, Canada

Short Abstract

To identify a suspected circulating non-functional ACTH isoform in an individual with a silent corticotroph adenoma, we developed a differential mass spectrometry (dMS) and targeted MS/MS approach. For dMS experiments, we compared ACTH immunoprecipitated from the individual in question, to the plasma from individuals with elevated ACTH and typical manifestations of ACTH excess. MS experiments revealed the presence of a truncated form of ACTH and MS/MS was used to confirm the identity of both the wild-type and aberrantly processed ACTH isoform. This dMS—MS/MS approach has the potential to be applied broadly in cases of structurally abnormal ACTH production.

Long Abstract

INTRODUCTION. A lesion in the left parasellar region was identified in a 64-year-old male (“patient”). Immunohistochemical studies of a biopsy of the tumor demonstrated immunopositivity for ACTH. Subsequent laboratory investigations revealed consistently elevated plasma ACTH, normal cortisol, and no signs or symptoms typically associated with elevated ACTH. To reconcile immunoassay, immunohistochemistry and clinical findings, we employed differential mass spectrometry (dMS) and targeted MS/MS to identify a suspected circulating non-functional ACTH isoform(s).

METHODS. Plasma ACTH was analyzed using two commercial immunoassays platforms: Siemens Immulite 2000 and Roche Cobas e602. Analysis included 5 plasma specimens collected from the patient over a 3-year period and 8 “control” plasma specimens with elevated ACTH. ACTH was immunoprecipitated using antibody-bound magnetic particles. The eluate was dried by vacuum centrifugation and reconstituted for liquid chromatography-MS analysis. To identify abnormal ACTH isoforms, precursor ion intensities from the patient sample were compared against spectra from control samples. MS/MS was used to confirm precursor ion identities. Data was acquired using a C18 analytical column coupled to an electrospray ionization quadrupole mass spectrometer (SCIEX 5500 QTRAP) and a linear MALDI-TOF mass spectrometer (Shimadzu AXIMA Assurance).

RESULTS. MS/MS analysis confirmed the presence of wild-type ACTH (residues 1-39, MH+5 = 908.7 m/z) in both patient and control samples. Comparative analysis of mass spectrometric data from patient and control specimens was used to identify the presence of mis-processed ACTH isoforms.

CONCLUSIONS. We developed an immunoprecipitation protocol to purify femtomole quantities of ACTH from human plasma to enable dMS and MS/MS characterization. This approach identified an ACTH isoform present in the patient’s specimen that is consistent with the clinical picture (silent corticotroph adenoma) and laboratory findings (cross-reactivity with immunoassays). Additional high-resolution/accurate mass experiments are underway to confirm these findings.

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