High-throughput matrix-assisted laser desorption ionization-time-of-flight mass spectrometry method for quantification of hepcidin in human urine.
Levels of the peptide hormone hepcidin negatively correlate with systemic iron status and are increased in disorders in which iron metabolism is secondarily disregulated, such as the anemia of chronic disease. Consequently, the ability to measure hepcidin in the clinical setting may have diagnostic value for a broad range of indications. We describe a novel quantitative matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry assay for hepcidin in human urine which involves (i) direct enrichment from minute volumes (5 microL) of minimally treated urine on the surface of a functionalized chip, (ii) quantification by the use of a stable isotope labeled internal standard, and (iii) analysis by MALDI-TOF. Performance features include a wide linear range (1-1000 nM; LOQ 2.5 nM), high accuracy (90-110% recovery) and precision (intraday CV 12.11%; interday CV 13.21%), and a strong correlation upon interlaboratory cross validation with an existing immunoassay. The assay is simple, accurate, and efficient, and the high-throughput performance features of the assay make large-scale clinical research studies feasible.
Anderson
Damon S
DS
Department of Pathology, Children's Hospital Boston, Boston, Massachusetts 02115, USA.
Heeney
Matthew M
MM
Roth
Udo
U
Menzel
Christoph
C
Fleming
Mark D
MD
Steen
Hanno
H
D023181:Antimicrobial Cationic Peptides / Q000652:urine*; D064451:Hepcidins; D057166:High-Throughput Screening Assays; D006801:Humans; D007118:Immunoassay; D016014:Linear Models; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D016482:Urinalysis / Q000379:methods*
D023181:Antimicrobial Cationic Peptides; C578702:HAMP protein, human; D064451:Hepcidins