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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 (13)C(15)N adenosine isotope dilution, internal (13)C(15)N 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.

The endogenous peptides of human serum may have regulatory functions, have been associated with physiological states, and their modifications may reveal some mechanisms of disease. In order to correlate levels of specific peptides with disease alongside internal standards, the polypeptides must first be reliably extracted and identified. Endogenous blood peptides can be effectively enriched by precipitation of the serum with organic solvents followed by selective extraction of peptides using aqueous solutions modified with organic solvents. Polypeptides on filter paper were assayed with Coomasie brilliant blue binding. The polypeptides were resolved by detergent tricine polyacrylamide electrophoresis and visualized by diamine silver staining. Peptides in the extracts were collected by C18 and analyzed by matrix-assisted laser desorption/ionization and liquid chromatography-electrospray ionization-tandem mass spectrometry (MS/MS) quadrupole time-of-flight MS/MS. Peptides were resolved as multiple isotopic peaks in MS mode with mass deviation of 0.1 Da or less and similar accuracy for fragments. The sensitivity of MS and MS/MS analysis was estimated to be in the picomolar range or less. The peptide composition of the extracts was dependent on solvent formulation. Multiple peptides from apolipoproteins, complement proteins, coagulation factors, and many others were identified by X!Tandem with high mass accuracy of peptide ions and fragments from collision-induced dissociation. Many previously unreported posttranslational modifications of peptides including phosphorylations, oxidations, glycosylations, and others were detected with high mass accuracy and may be of clinical importance. About 4,630 redundant peptides were identified with 99% confidence separately, and together some 1,251 distinct proteins were identified with 99% confidence or greater using the Paragon algorithm.