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Sep 26, 2014 - The specificity and amplification of the signal by the enzyme alkaline phosphatase in ... Angelique Florentinus-Mefailoski , John G. Ma...
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Pyridoxamine-5-phosphate Enzyme-Linked Immune Mass Spectrometric Assay Substrate for Linear Absolute Quantification of Alkaline Phosphatase to the Yoctomole Range Applied to Prostate Specific Antigen Angelique Florentinus-Mefailoski and John G. Marshall* Department of Chemistry and Biology, Ryerson University, 350 Victoria Street, Toronto, Ontario, Canada, M5B 2K3 S Supporting Information *

ABSTRACT: There is a need to measure proteins that are present in concentrations below the detection limits of existing colorimetric approaches with enzyme-linked immunoabsorbent assays (ELISA). The powerful enzyme alkaline phosphatase conjugated to the highly specific bacterial protein streptavidin binds to biotinylated macromolecules like proteins, antibodies, or other ligands and receptors with a high affinity. The binding of the biotinylated detection antibody, with resulting amplification of the signal by the catalytic production of reporter molecules, is key to the sensitivity of ELISA. The specificity and amplification of the signal by the enzyme alkaline phosphatase in ELISA together with the sensitivity of liquid chromatography electrospray ionization and mass spectrometry (LC−ESI-MS) to detect femtomole to picomole amounts of reporter molecules results in an ultrasensitive enzyme-linked immune mass spectrometric assay (ELIMSA). The novel ELIMSA substrate pyridoxamine-5-phosphate (PA5P) is cleaved by the enzyme alkaline phosphatase to yield the basic and hydrophilic product pyridoxamine (PA) that elutes rapidly with symmetrical peaks and a flat baseline. Pyridoxamine (PA) and 13C PA were both observed to show a linear relationship between log ion intensity and quantity from picomole to femtomole amounts by liquid chromatography−electrospray ionization and mass spectrometry. Four independent methods, (i) internal 13C isotope PA dilution curves, (ii) internal 13C isotope one-point calibration, (iii) external PA standard curve, and (iv) external 13C PA standard curve, all agreed within 1 digit in the same order of magnitude on the linear quantification of PA. Hence, a mass spectrometer can be used to robustly detect 526 ymol of the alkaline phosphatase streptavidin probe and accurately quantify zeptomole amounts of PSA against log linear absolute standard by micro electrospray on a simple ion trap.

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INTRODUCTION Enzyme-Linked Immune Mass Spectrometric Assay (ELIMSA) for Low-Abundance Analytes. Human blood contains low-abundance proteins of biomedical interest at concentrations often below the detection limits of either enzyme-linked immunoabsorbent assays (ELISA)1 or mass spectrometry2 when used separately. Combining the enzymatic amplification of ELISA with the detection of the enzymatic products by liquid chromatography, electrospray ionization and mass spectrometry (LC−ESI-MS) results in the ultrasensitive quantification method termed ELIMSA,3 potentially allowing the routine analysis of femtogram amounts of low-abundance proteins in solution. The enzymatic amplification is obtained from alkaline phosphatase (AP), a near perfect enzyme4 that may be attached to the bacterial protein streptavidin (SA). Streptavidin conjugated5 to the reporter enzyme (AP-SA) may be used to detect biotinylated antibodies, ligands, or probes with a high affinity.6 Enzymatic reactions have been previously monitored by mass spectrometry,7 and the substrate pyridoxamine-5-phosphate (PA5P) has been used to generate an © 2014 American Chemical Society

ionizable product by alkaline phosphatase for ion mobility spectrometry.8 Moreover, mass spectrometry has been used to confirm the nature of the products from an ELISA reaction.9 Hence, there is good reason to believe it should be possible to implement ELIMSA using the substrate PA5P that releases pyridoxamine.10 Prostate Specific Antigen (PSA) Model System. PSA is not selective for malignant disease but is a tissue-specific kallikrein protease that indicates benign and cancerous proliferation of prostate tissue and cells.11 The prostate specific antigen (PSA) has a concentration distribution from picogram to nanogram amounts in 100 μL of normal human plasma (NHP) from men, and so like many cytokines and regulatory factors it is often near or below UV−vis detection in normal patients.12 The quantification of PSA over the complete range of NHP is challenging by ELISA12 or mass spectral methods,2 Received: July 12, 2014 Accepted: September 26, 2014 Published: September 26, 2014 10684

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The alkaline phosphatase-streptavidin (AP-SA) enzyme-probe conjugate was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). The liquid chromatography− mass spectrometry grade solvents were obtained from Caledon Laboratories (Georgetown, Ontario, Canada). Immunoassay. The PSA ELIMSA was performed as previously described but with the substitution of the substrate PA5P instead of naphthol AS-MX phosphate.3 The PA5P substrate, PSA standards and AP-SA probe were dissolved in water on ice, aliquoted, and freeze-dried for day-to-day consistency in the assay. The plates were coated with the capture antibody in bicarbonate coating buffer prior to blocking the plates in 1% albumin and 1% goat serum in 1× PBS pH 7.4 followed by incubating the plasma in 1× PBS, 0.3 M NaCl pH 7.4 for 2 h. After the wells were washed again three times with washing buffer, the substrate was reacted with the streptavidin− AP in substrate buffer (20 mM Tris pH 8.8). The experimental assay was first optimized and tested using large amounts of PSA with colorimetric reagents to ensure there is no background binding and to confirm the antibodies and calibrants are working and linear in color assays before switching to ELIMSA to measure the lower standards and concentrations for normal human plasma (NHP). Liquid Chromatography−Electrospray IonizationMass Spectrometry. The LC-ESI-MS analysis was performed by manual injection (Rheodyne 1755) of 2 μL of the standard or sample diluted 20-fold in 0.1% formic acid. The sample was analyzed via an Agilent 1100 high-pressure liquid chromatography (HPLC) pump set at 70% acetonitrile in 0.1% acetic acid for isocratic separation over a 15 cm normal phase column (300 μm i.d., 5 μm particle diameter, 300 Å pore) at 20 μL/min. We compared normal phase and C18 reversed phase columns. Although a C18 gradient provided sharper peaks, it was not as convenient as the rapid isocratic normal phase column. The normal phase chromatogram produced symmetrical peaks that were resolved from the buffer components with low back pressure and high flow rates allowing rapid analysis with baseline to baseline separation over a stable background. The HPLC was coupled via an electrospray source fitted with a metal needle17 mounted on a linear quadruple ion trap (LTQ XL, Thermo Electron Corporation)18 as previously described.3 The PA5P [M + H] product ion was monitored by intensity at 169 m/z as previously described3 with the injection of 70% IPA via the sample loop to rapidly clean the column between injections. The instrument was tuned and the correct calibration of the product m/z assured using the manufacturer’s tuning solution and the PA product. The intensity of the pyridoxamine (PA) was monitored by selected ion monitoring (SIM) of the window 168−170 m/z where the intensity of the [M + H] ion at 169 m/z was extracted and plotted. The intensity of the 13C PA standard was monitored by the SIM window 171−173 m/z where the intensity of the [M + H] ion at 172 m/z was extracted and plotted. The results were logtransformed, and the ion intensity results were analyzed for normality and linearity16b using the open source R statistical analysis system.

and many normal samples are below the detection limit of the assay. Detection of an increase in baseline PSA over time in normal or prostatectomy patients may be of clinical importance in some cases. Many other tissue or cell specific proteins, regulatory factors, and cytokines remain to reliably assay in blood.13 There are other good experimental reasons to consider PSA as a test protein for developing more sensitive assays such as ELIMSA: the analyte PSA and the enzyme AP-SA have been used to benchmark the sensitivities of ELISA,14 direct mass spectral detection,2 and electrochemical detection15 as well as a previous ELIMSA substrate.3 The PSA ELISA using the high affinity reagents described here is an excellent model system for demonstrating the principles of ELIMSA and testing substrates. Linearity and Accuracy of LC−ESI-MS. It has been previously shown that an ELISA with the colorimetric enzyme substrate amplex red, and naphthol AS-MX phosphate, can be measured by LC−ESI-MS with linearity similar to that of UV− vis based detection.3 Here, the same specific PSA calibrant, capture, and detection reagents used previously were tested with the novel ELIMSA substrate PA5P.3 It remains to be determined if the enzymatic product PA shows a predictable relationship between intensity and quantity in the context of the immune assay. A linear relationship between log ion intensity and quantity was observed for some blood peptides or fragment ions, peptide standards, resorufin, naphthol AS-MX, and in the present experiment pyridoxamine (PA) that were all shown to be linear after simple log transformation.3,16 Internal isotopic labeled standards may be used to test the assumptions about the linearity and accuracy of the LC−ESI-MS assay. Advantages of PA5P Substrate. In this paper, we show that the substrate pyridoxamine-5-phosphate (PA5P) has several important practical advantages over the previous ELIMSA substrate naphthol AS-MX phosphate in that the enzyme product pyridoxamine (PA) is basic, ionizes readily but is also hydrophilic and elutes rapidly with a more symmetrical peak. Most importantly, PA5P shows a more predictable chromatography, is not strongly retained, and does not show a creeping increase in baseline over the course of measurement. The use of PA5P has an important additional advantage in that the enzyme product PA, sometimes termed vitamin B6, is a common metabolite that is widely available in 13C isotope labeled form that can be used as an internal standard to confirm the absolute quantification of PA for ELIMSA. Thus, to address the issue of transformation, linearity, and absolute quantification in ELIMSA by LC−ESI-MS, we controlled the ELIMSA experiments with internal, and external, 13C labeled pyridoxamine (13C PA) standards to confirm the quantification of the enzyme product. If mass spectrometry is log linear and normal for PA, then instead of using UV−vis spectroscopy to read the 96 well ELISA plate, the production of the colorless PA could be measured using LC−ESI-MS with a sensitive ion trap.3 The capacity to measure zeptomole amounts of blood proteins may have great application to biomedical research and permits the common analysis of many ultra low abundance analytes.

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MATERIALS AND METHODS Materials. The NHS-biotin coupling reagent was obtained from Pierce. The PSA capture and detection antibodies were purchased from Medix Biochemica (Kauniainen, Finland). The PSA calibration antigen was obtained from the Scripps Laboratory (San Diego, CA). The PA5P, pyridoxamine, PA, and 13C (PA), and all other buffers and salts were obtained from Sigma-Aldrich and were of the highest quality available.

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RESULTS Pyridoxamine Has Excellent Chromatography Characteristics. Previously, the alkaline phosphatase (AP) enzyme system was more sensitive than the HRP system that requires the strong oxidizing agent H2O2 as a cofactor,3 and so the development of the AP based ELIMSA was continued by 10685

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Figure 1. Comparison of the PA and 13C PA external standards by LC−ESI-MS. Panels: (A) the isocratic chromatogram showing the dilution series of PA [M + H] monitored at SIM 169 m/z (inset shows linearity after log transformation); (B) the isocratic chromatogram showing dilution series of 13C PA monitored at SIM 172 [M + H] m/z (inset shows agreement between natural and isotope-labeled 13Cpyridoxamine). The isocratic chromatograms show the injection of 2 μL of the standard in nanomolar as indicated by the arrows in 0.1% FA on normal phase in 70% acetonitrile connected to an electrospray source.

Figure 2. Enzymatic production of pyridoxamine (PA) from the substrate pyridoxamine-5-phosphate (PA5P) by alkaline phosphatase conjugated to streptavidin (AP-SA). Panels: (A) normal phase isocratic chromatogram of the time course of PA production by the AP-SA conjugate (5 ng) added to 1 mL of 1 mM PA5P in 20 mM tris pH 8.8 as measured using LC−ESI-MS with SIM of pyridoxamine (PA) at 169 m/z [M + H] where the arrow shows blank (b), no enzyme N.E., or the enzyme reaction time in minutes (inset shows the plot of log 169 [M + H] intensity versus time); (B) AP-SA conjugate amount shown by the arrow in nanograms was added to 1 mL of 1 mM PA5P in 20 mM tris pH 8.8 (inset shows the log linear relationship between log PA ion intensity when the amounts of AP-SA in log ng/mL were added to the reaction). The production of PA was sampled by removing 10 μL of the reaction that was mixed with 190 μL of 0.1% formic acid and 2 μL of the diluted sample was manually injected. Blank injection (b) and no enzyme (N.E.) reaction injections are shown where indicated.

looking for more effective substrates. The use of the alkaline phosphatase substrate PA5P was found to be a strong improvement on the previous substrate naphthol AS-MX phosphate in that the latter’s hydrophobic naphthol substituent causes distinct tailing in the chromatographic peaks with the result that baseline to baseline separation is not always obtained at convenient injection intervals (≤5 min.) significantly increasing analysis time. In contrast, the basic and hydrophilic product pyridoxamine (PA) ionizes readily, shows much more symmetrical peaks and easily achieves baseline to baseline separation even at close injection intervals (see the Supporting Information). Pyridoxamine Ion Intensity Is Log Linear with Quantity. The ELIMSA assay for PSA depends on the production of PA that is measured by LC−ESI-MS and so it is necessary to demonstrate that the relationship between PA quantity and signal intensity is log linear and predictable. A simple dilution curve of pyridoxamine (PA) in ionization buffer reveals a log linear relationship between intensity and quantity when injected into the isocratic LC−ESI-MS system. The PA [M + H] product ion showed a log linear relationship between ion intensity versus absolute quantity as measured using LC− ESI-MS with single ion monitoring (SIM) at 169 m/z (Figure

1A). Similarly, the 13C PA standard parent ion [M + H] at SIM 172 m/z also showed a log linear relationship between ion intensity and quantity (Figure 1B). However, since electrospray ionization is a competitive process, the linearity of PA in pure ionization buffer may not be the same as the ionization of PA in the ELIMSA reaction where there may be some complex confounding effects of the tris buffer, blocking agents, nonionic detergents, or other buffer components. Enzyme Dependent Release of Pyridoxamine by Alkaline Phosphatase-Streptavidin. The ELIMSA assay depends on the amplification of the signal from the AP-SA probe by the enzymatic activity of alkaline phosphatase. If the PA signal is generated by the AP-SA probe then it should be dependent on enzyme incubation time and the enzyme concentration. Adding the biotin-specific molecular probe streptavidin, conjugated to the enzyme AP-SA, to the substrate 10686

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Figure 3. Illustration of the experimental setup to measure ELIMSA with absolute external PSA standards by the pyridoxamine (PA) production from the AP-SA probe alongside internal isotope dilution, internal one-point calibration, and external PA and 13C PA standard curves. Panels: (A) internal 13C isotope dilution curve for 12 NHP samples (left axis) that showed an average of ∼2.01 ± 0.0377 pmol of PA injected from each replicate well (right axis); (B) isocratic chromatogram showing the ELIMSA with an external PSA standard curve that was linear from 0.1 to 10 ng per well with 1.5 pmol 13C internal standards, followed by 10 NHP samples with 1.5 pmol 13C internal standards, and finally PA and 13C PA external standards as shown; (C) main panel shows the average PSA standard intensity values over 3 separate days with an R2 of 0.99 (insets: upper left, the external pyridoxamine (PA) standard curve with SIM 169 [m/z] and the 13C external PA curve SIM 172 m/z; lower right, the intensity match between the NHP PSA ELIMSA at SIM 169 and 1.5 pmol of the 13C pyridoxamine internal standard that showed a nearly equal intensity match between the NHP PSA ELISA at SIM 169 and 1.5 pmol of 13C PA internal one-point calibration standard).

was PA [M + H] produced by the enzymatic dephosphorylation of the substrate PA5P by the AP-SA probe. Estimate of PA Production from ELIMSA by Internal 13 C PA Dilution. The intensity values of the LC−ESI-MS assay for PA remained linear in the context of the ELIMSA biochemical reaction conditions (Figure 3), and the internal isotope dilution method was used to estimate the quantity of internal 13C labeled PA that matched the average production of PA from the ELIMSA. The internal isotope dilution method was used to estimate the average production of PA from the same replicate NHP sample (Table 1) and was found to be about 2.01 pmol of PA measured against the internal 13C PA internal isotope dilution curve (Figure 3A, Table 1).

PA5P in tris reaction buffer released the product PA and the biochemical reaction was essentially complete by 3 h (Figure 2A). The time course of the AP-SA signal closely matched an expected enzyme activity with an initial nearly linear period of rapid production followed by an asymptotic decrease in slope as the reaction approached completion. The AP-streptavidin conjugate (AP-SA ∼190 000 g per mole) was diluted down to 0.1 ng per mL and then the enzyme was reacted with the substrate PA5P for 3 h to produce PA. After diluting the reaction 20-fold and injecting 2 μL, i.e., the equivalent of 0.1 μL of the reaction, about 50 zmol of AP-SA on column was detected. The enzyme dependent production of PA was clearly detectable with a high signal-to-noise ratio and low error in the baseline (Figure 2B). The pattern with time and concentration both seemed to indicate that the signal measured at 169 m/z 10687

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Table 1. Agreement between Internal 13C PA Isotope Dilution, Internal One-Point 13C PA Calibration, External PA Standard Curve and External 13C PA Standard Curve, Alongside the Results of the External PSA Standard Curve Applied to Selected NHP Samplesa method

result

C PA isotope dilution PA external curve 13 C external curve 13 C PA one-point internal calibration PSA external curve

2.01 pmol PA 2.19 pmol PA 1.58 pmol PA 1.28 pmol PA 57.78 pg PSA/well

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ion [M + H] monitored at SIM 169 m/z against the external standard 13C PA monitored at 172 m/z showed 1.58 and 2.19 of pmol of PA on column in agreement with internal isotope dilution and internal one-point calibration (Figure 3C, Table 1). Thus, when on the order of 1 amol of PSA standard was analyzed, the ELIMSA reaction produced on the order of 1 pmol amounts of PA and thus apparently achieved nearly a million-fold amplification (Table 1). ELIMSA for Prostate Specific Antigen from Normal Human Plasma. ELIMSA was applied to measure PSA in NHP that was preselected to be below the detection limit of the colorimetric assay as a test of the system under authentic conditions. The external PSA standard showed a log linear relationship between PA [M + H] ion intensity versus PSA quantity and good technical reproducibility as measured by replicate analysis of test samples using LC−ESI-MS with single ion monitoring (SIM) at 169 m/z (Figure 3C). A total of 10 randomly selected normal plasma (NHP) samples were analyzed on 3 independent days in a row with good agreement and fitted to the calculated normal distribution. The average amount of PSA in the NHP samples was about 67.8 pg with a standard error of about 4.3 pg from a total of 30 NHP samples (50 μL) (Table 2). The estimated PSA values showed a Guassian, i.e., normal distribution, by the Shapiro-Wilk test (N = 30, W 0.968499 Prob < W 0.3222) and the plotted distribution showed little deviation from the normal curve as illustrated with the quantile plot produced with the statistical analysis system R (see the Supporting Information). In the application of ELIMSA to measure PSA in the preselected NHP samples that were undetectable by color assays, the results were all 33 pg per well or greater and were within the limits of the known standards for detection and quantification by ELIMSA. Since all of the normal male plasma samples were within the limit of quantification of the assay, it was not possible to establish the lower limit of ELIMSA for PSA in patient samples. Ultrasensitive Detection. In order to estimate the lowest possible detection of the AP-SA enzyme conjugate, a dilution series of AP-SA was made that showed a clear detection of 1 pg/mL as quantified by the injection of 0.1 μL (∼526 ymol of AP-SA on column) at 20 μL per minute with robust electrospray and a simple ion trap (Figure 4). The contamination of the SIM 169 m/z channel by some minor component in the injection buffer is especially obvious at low concentrations. Hence, it was possible to clearly detect the presence of ∼1 fg of AP-SA on column or less at 20 μL per minute through an electrospray source with a simple ion trap.

a

The normal human plasma (NHP) samples were pre-selected from samples that failed to yield colorimetric values. The levels of prostate specific antigen (PSA) in selected normal human plasma samples (NHP) was estimated from an external standard curve of absolute PSA amounts using an LC−ESI-MS assay for pyridoxamine produced by the enzyme conjugated AP-SA probe that was bound to the biotinylated PSA detection antibody. The NHP sample was selected from those that had less than 0.1 ng of PSA per 100 μL and so could not be measured by colorimetric methods and was aliquoted for replicate experimentation. The experimental design of the analytical chromatography experiments used to address absolute quantification of PSA by an LC−ESI-MS assay to provide these values of PA is shown in Figure 3. The results indicate that about 2 pmol of PA is produced when about 3 amol of PSA is provided and so shows about a million fold amplification of the signal.

Table 2. Replication of the Analysis in Normal Human Plasma That Was Selected As Too Low to Estimate PSA by the Colorimetric Assay Is Shown in pg PS/well)a

mean Std Err.

day 1

day 2

day 3

93.04 58.30 61.28 83.35 41.06 63.34 65.46 50.10 67.64 84.02

105.33 58.13 42.26 41.88 35.33 33.75 89.46 75.67 85.32 73.26

58.36 61.30 68.37 77.90 48.96 61.30 82.59 102.53 88.51 75.20

66.67 5.07

64.04 8.01

72.5 5.06

all 3 days 67.76 4.32

a

The within day mean and standard error (Std Err.) is shown vertically below and the summary over all 3 days to the right.

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DISCUSSION Sensitivity. Direct analysis of the generic detection reagent AP-SA by LC−ESI-MS permits the quantification of the AP-SA probe to at least 1 pg per mL range from the injection of 0.1 μL from the sample (i.e., yoctomole amounts of the molecular probe on column) and was sufficiently sensitive to detect all the low-abundance PSA samples in this study. The sensitivity was such that the PSA values of all NHP were well within the standards at concentrations far below the robust detection limit of color, fluorescence, or ECL methods.3 The ELIMSA assay seems to detect the presence of the probe to levels comparable to or beyond that of radio immunometric assays but without the administrative burden of handling radioisotopes. The sensitivity of the assay is limited at least in part by confounding substances in the injection buffer and the background binding

The amount of PA produced by the ELIMSA reactions was also estimated by the one-point calibration against the addition of 1.5 pmol of 13C PA added to each NHP ELIMSA reaction sample. Each replicate ELIMSA reaction was monitored for PA [M + H] at SIM 169 m/z alongside 1.50 pmol of the 13C PA [M + H] internal standard monitored at SIM 172 m/z (Figure 3B, Table 1). There was good agreement between the independent internal isotope dilution curve and the one-point ratio estimate from a pooled normal sample that showed about 1.28 pmol of PA. On the same day, and on the same chromatogram as the one-point calibration experiment, we also measured PA production in terms of external 13C PA and PA standard curves with the analysis of 0.1 μL analyzed from each 100 μL well. Reading the amount of pyridoxamine (PA) produced by the ELIMSA of NHP plasma from the molecular 10688

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Figure 4. Detection of AP-SA from 1 to 100 pg per mL with analysis of 0.1 μL that shows sensitivity on the order of 5.26 × 10−22 mol, i.e., 526 ymol or ∼1 zmol, of AP-SA on column.

PA internal isotope dilution curve. Together, these several controls unambiguously demonstrate that ELIMSA of PSA is based on LC−ESI-MS assays that are log linear and accurate. The PA and 13C PA ion intensity values were linear after log transformation, and the calculated PSA results were normal and so suitable for analysis of variance (ANOVA) statistical analysis between experimental treatments. However, since the PSA standard data may have to be log transformed and so cannot pass through zero, it is traditional to calculate the intensity or quantity of the data as log (i + 1). After log transformation, the data apparently shows acceptable linearity over at least 3 orders of magnitude (R2 of ∼0.99). Moreover, the results of PSA in the low-abundance normal human plasma by ELIMSA here closely match the correct order of magnitude for PSA reported in the literature. Thus, multiple lines of evidence in agreement indicate that the ELIMSA, standardized against known amounts of the PSA calibrant, provides the accurate absolute quantification of PSA. It was previously demonstrated that ELISA and ELIMSA gave results that were linear and consistent in the nanogram per well range by direct comparison to colorimetric assays.3 It seems that ELIMSA may be used to absolutely quantify PSA proteins at picograms per well. Since all the normal samples analyzed here were no less than 33 pg per well, all samples were well within the quantitative range of the assay as measured against absolute PSA standards. Log linearity of LC−ESI-MS. In this study, the external PSA curve, the internal 13C PA isotope dilution curve, the external PA curve, and the external 13C PA isotope standard curves all agreed that mass spectrometry showed a linear relationship with quantity after simple log transformation. The log linear results with PA were in agreement with log linear

of the biotinylated enzyme probe to the ELISA plate that must be accounted for in the calculations. Sensitivity will vary on a case by case basis depending on the quality of available reagents. However, the capacity of ELIMSA to quantify molecules at low concentrations is so great that the sensitivity of the detection method will no longer be the limiting factor in most experiments. Agreement between Independent Methods. The result with the internal 13C isotope dilution curve was in close agreement with the external PA curve and the external 13C PA curve. The one-point calibration method showed the greatest divergence, but four independent methods were within 1 digit in the same order of magnitude and so in general showed good agreement. The results of ELIMSA to quantify PSA in the colorimetric range provided results that closely matched the results of well-established quantification using the amplex red and naphthol AS-MX phosphate substrates. Hence, there are multiple lines of evidence in agreement that LC−ESI-MS may provide for the accurate quantification of molecules such as PA after log transformation. The internal isotope assays here required the assumption of log linearity and agreed with the external curves that were also suitably linear after log transformation. No complex mathematics of any kind were required to solve for the values presented, and the log linear regression of the signals and quantification was robust. Absolute Quantification of PSA by ELIMSA. The ELIMSA employs an external absolute PSA standard dilution curve monitored from LC−ESI-MS of the PA released by the AP-SA probe. The linear results with the authentic PSA protein used as an external, absolute standard were consistent with the linearity of PA and 13C PA external standard curves and the 13C 10689

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Author Contributions

results from some standard peptides or peptides from blood proteins,16a resorufin, and naphthol AS-MX,3 many peptides and proteins from cells,16c or proteins secreted from cells into the experimental media.16b Taken together, the results with small molecules, isotopic labels, and many peptides all indicate that LC−ESI-MS is apparently log linear for some pure compounds and thus may approach linearity for some molecules after preseparation by chromatography. Linear signal intensities that are only a small fraction of the total ion current are consistent with the competitive nature of the electrospray ionization process19 in which the presence of other analytes may affect observed intensities in unpredictable ways. The potentially confounding effects of competition for ionization were apparently not a factor in this case in which relatively pure analyte PA dissolved in a 1 mM tris buffer is efficiently resolved by high-pressure liquid chromatography prior to ionization with results closely approaching log linearity.

A.F.-M. screened the substrates, optimized the detection conditions, performed most experiments and numerical analysis, created the charts and graphs, and proofed the manuscript. J.G.M. performed preliminary experiments, conceived and designed the ELIMSA experiment, wrote the proposal, performed the statistical analysis, created the final figures, and wrote the paper. Notes

The authors declare no competing financial interest.

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

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CONCLUSION The use of LC−ESI-MS to measure an enzyme activity with a conjugated molecular probe provides a universal system for the quantification of macromolecules including proteins, ligands, and/or receptors at femtogram to picogram amounts or less with good signal-to-noise values. The detection of yoctomoles of the AP-SA probe and measurement of zeptomoles of PSA is comparable to, or more sensitive than, other methods claimed to date2,14,15,20 but requires only commonly available micro electrospray and a simple ion trap. ELIMSA with the substrate PA5P that releases pyridoxamine showed more symmetrical peaks, baseline to baseline separation even at close injection intervals, and a much flatter baseline compared to the previous substrate naphthol AS-MX phosphate and was a major improvement to the method. The experiments here with external and internal standards and isotope dilutions clearly demonstrate that LC−ESI-MS may provide a linear relationship between intensity and quantity after log transformation. All of the PSA samples in this study were within the robust quantification range of the ELIMSA in the zeptomole range on column and could be estimated by interpolation between known amounts of PSA standard solutions that were log linear with a high signal-to-noise ratio. Here pyridoxamine, AP-SA, and PSA molecules were absolutely quantified by LC−ESI-MS against external standards and the linearity and accuracy of the PA was supported by the use of internal isotopic standards. The log linearity and normality of mass spectral intensity after log transformation opens up broad vistas for the direct quantification of many molecules by classical linear models and ANOVA statistical analysis.3,16 The experiments here unambiguously demonstrate that enzyme labeled probes can sensitively quantify proteins or receptor−ligand complexes to zeptomole amounts by direct mass spectrometric analysis and will have a broad application and utility for measuring molecules at low concentrations that greatly extends the practical sensitivity of ELISA biomolecular analysis.

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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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