Analytical and Bioanalytical Chemistry

, Volume 407, Issue 4, pp 1119–1130 | Cite as

An enzyme-linked immuno-mass spectrometric assay with the substrate adenosine monophosphate

  • Angelique Florentinus-Mefailoski
  • Antonius Soosaipillai
  • Jaimie Dufresne
  • Eleftherios P. Diamandis
  • John G. MarshallEmail author
Research Paper


An enzyme-linked immuno-mass spectrometric assay (ELIMSA) with the specific detection probe streptavidin conjugated to alkaline phosphatase catalyzed the production of adenosine from the substrate adenosine monophosphate (AMP) for sensitive quantification of prostate-specific antigen (PSA) by mass spectrometry. Adenosine ionized efficiently and was measured to the femtomole range by dilution and direct analysis with micro-liquid chromatography, electrospray ionization, and mass spectrometry (LC-ESI-MS). The LC-ESI-MS assay for adenosine production was shown to be linear and accurate using internal 13C15N adenosine isotope dilution, internal 13C15N adenosine one-point calibration, and external adenosine standard curves with close agreement. The detection limits of LC-ESI-MS for alkaline phosphatase–streptavidin (AP-SA, ∼190,000 Da) was tested by injecting 0.1 μl of a 1 pg/ml solution, i.e., 100 attograms or 526 yoctomole (5.26E−22) of the alkaline-phosphatase labeled probe on column (about 315 AP-SA molecules). The ELIMSA for PSA was linear and showed strong signals across the picogram per milliliter range and could robustly detect PSA from all of the prostatectomy patients and all of the female plasma samples that ranged as low as 70 pg/ml with strong signals well separated from the background and well within the limit of quantification of the AP-SA probe. The results of the ELIMSA assay for PSA are normal and homogenous when independently replicated with a fresh standard over multiple days, and intra and inter diem assay variation was less than 10 % of the mean. In a blind comparison, ELIMSA showed excellent agreement with, but was more sensitive than, the present gold standard commercial fluorescent ELISA, or ECL-based detection, of PSA from normal and prostatectomy samples, respectively.


ELISA Liquid chromatography Electrospray Mass spectrometry Adenosine monophosphate Alkaline phosphatase ELIMSA 



Adenosine monophosphate


Alkaline phosphatase–streptavidin


Enhanced chemiluminescence


Enzyme-linked immunosorbent assay


Enzyme-linked mass spectrometric assay


Liquid chromatography electrospray ionization and mass spectrometry


Prostate-specific antigen


Single ion monitoring



This work was supported by a Discovery Grant from the Natural Science and Engineering Research Council of Canada to JGM.

Supplementary material

216_2014_8323_MOESM1_ESM.pdf (705 kb)
ESM 1 (PDF 704 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Angelique Florentinus-Mefailoski
    • 1
  • Antonius Soosaipillai
    • 3
  • Jaimie Dufresne
    • 1
  • Eleftherios P. Diamandis
    • 2
    • 3
    • 4
  • John G. Marshall
    • 1
    Email author
  1. 1.Department of Chemistry and BiologyRyerson UniversityTorontoCanada
  2. 2.Department of Laboratory Medicine and PathobiologyUniversity of TorontoTorontoCanada
  3. 3.Department of Pathology and Laboratory MedicineMount Sinai HospitalTorontoCanada
  4. 4.Department of Clinical BiochemistryUniversity Health Network and Toronto Medical LaboratoriesTorontoCanada

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