This paper reports an application of the SAMDI-TOF MS method for performing immunoassays of protein markers in clinical samples. A body of previous work has demonstrated that SAMDI is well suited to the analysis of low molecular weight species, including peptides, carbohydrates and organic molecules. Indeed, SAMDI has been used to perform a range of enzyme activity assays—including kinase, protease, methyltransferase and glycosyltransferase activities—and has been applied to screening small molecule libraries to identify antagonists of enzyme activities.35, 49, 50, 52, 68
The extension of SAMDI from detecting low molecular weight species to large molecular weight proteins, and from analyzing solutions of defined and relatively simple composition to complex samples derived from bodily fluids, has not been previously demonstrated. A first report showed that SAMDI could observe molecular ions corresponding to intact proteins with masses up to approximately 50 kDa.46
That work used surfaces presenting glutathione ligands to immobilize GST-fusion proteins, with the limitation that the rate constant for dissociation of the GST-glutathione interaction limited the stability of the biochip in diagnostic applications. The current work is significant because it provides an additional example showing that SAMDI can analyze large molecular weight protein analytes (up to 150 kDa) and provides the first example that our monolayer surfaces are able to discriminate protein antigen(s) from the other components in complex samples. The previous examples from out group and this current paper highlights a significant benefit of this surface chemistry approach (over SELDI and traditional affinity biochip formats combined with mass spectrometry)69
in that it can be applied to a broad range of bioassays, now including those directed towards antigen detection, protein-protein interactions, and permitting enzyme activity assays on immobilized peptides, carbohydrates, and small molecules.
Several considerations prompted our use of the receptor protein G (or A) to immobilize the antibody to the monolayer. In a previous report that used cutinase-mediated immobilization to give oriented immobilization of antibodies, we showed that control over both density and orientation could be harnessed to optimize the activity of the antibody.60
One limitation for this assay was the necessity for a recombinant cutinase-antibody reagent. The current approach translates the advantages of the cutinase system to the well known use of protein G (or A) to immobilize and orient an IgG antibody through its nonantigenic Fc region.28–30, 59
The protein G (or A)-based assay can be applied to a broad range of immunoassays, provided that an IgG type antibody with reasonable affinity and selectivity for the intended analyte and the receptor protein is available.70
Further, the assay format reported here does not require modification of either the antibody or the analyte. Many immunosensor schemes, for example require chemical or enzymatic modification of the Fc region of the antibody to introduce functionality needed for the immobilization, and additionally require labeling of either antibody or antigen to detect the latter. Because SAMDI can observe the antigen directly, labeling is not required. Unlike sandwich immunoassays, which require two antibodies with high affinity and non-overlapping epitopes on the antigen, the use of SAMDI abrogates the need for the second antibody and substantially streamlines the assay development cycle. Finally, we note that the use of SAMDI as the detection method also serves to verify the integrity of the biosensor, since this technique can verify that each of the intended components is present. This benefit is not of high importance in the development of individual immunoassays—which undergo a stringent assay optimization and validation—but can be very important in array-based experiments where it is not feasible to establish the fidelity of each assay.
Mass spectrometric methods offer a significant benefit over optical methods for label-free detection, including surface plasmon resonance spectroscopy. Because the optical methods measure changes in the refractive index of the medium near the biosensor surface, they do not discriminate between the intended analyte and species that contribute to the background signal. SAMDI, and other methods that use mass spectrometry, provide the masses of the species interacting with the sensor surface and therefore can more efficiently identify, and quantitate, the signal for the intended analyte even when there are significant levels of background species bound to the sensor surface. Further, the mass filtering that is inherent to mass spectrometry methods permits multiple assays to be performed simultaneously. In this work, we showed that four antigens in serum could be analyzed with an immunosensor that presented a mixture of antibodies specific to each analyte. The same experiment would be difficult to perform with fluorescently-labeled reagents because of spectral overlap of the fluorophores, and would be essentially impossible to perform with radiolabeled reagents.
Although mass spectrometry has the clear benefit of differentiating between intended analytes and background, with the current experimental design there is the potential for interference resulting from signal from the receptor protein G (or A) or the antibody. Further, the receptor proteins can immobilize antibodies present in the sample. Proper choice of either protein G (27 kDa) or protein A (48 kDa) can be effective for eliminating spectral overlap between the analyte and the sensor. We also note that the current SAMDI method used ion extraction parameters that were set to provide sufficient signal across a broad m/z
range (5000–150,000 m/z
) but were not optimized for resolution of each individual analyte. Further optimization of the delayed extraction parameters will give better resolution across narrow mass ranges limiting the chance for interferences.71
Finally, proper selection of matrix can have a profound effect on the resulting charge state distributions from the antigen, protein G/A, and the antibody. In this work we used both sinapinic acid (SA, 5 mg/mL in acetone) and α-cyano-4-hydroxycinnamic acid (CHCA, 7.5 mg/mL in acetone) as the energy absorbing matrices. In each case, approximately 0.3 μL of matrix was spotted by hand with a micropipette and allowed to diffuse across the monolayer. We usually observed signals across the entire surface, but noted a variation of signal intensity and some concentration of the non-covalently bound components at the matrix frontal edge. Typically, SA provided greater S/N for the m/z
> 50,000 portion of the spectrum, which offered a more pronounced signal for the antibodies. In these instances, a greater surface area was required for sampling because higher laser intensities were necessary to obtain the spectra, which follow previous observations.22
The CHCA matrix permitted the use of lower laser intensities and therefore provided prolonged sampling across a given area of the surface, while yielding good peak intensities for m/z
< 50,000 portion of the spectrum. Signal resulting from the loaded antibodies was sometimes suppressed with CHCA while that of protein A/G molecule and antigen could still be observed (see for HSA vs. for transferrin). This effect may be a response to the high density of antigen loaded across the surface that may interfere with desorption and ionization of the antibody. Alternatively, this observation could result from localized concentration of antigens to regions of the immunosensor that were manually sampled. Control over matrix and matrix deposition will not only have an effect on ion signal intensity and charge state distribution but may help to optimize resolution by providing a more uniform density of analyte across the entire surface. Future efforts should evaluate devices such as aerosol emitting tips or ink jet printers that regulate the deposition of matrix on the surface or harness methods that avoid the use of matrix if they can be adapted to high molecular weight analytes.72–75
Mass spectrometric methods are particularly useful for the characterization of proteins that contain metabolically-derived post-translational modifications or protein variants associated with gene families (or single nucleotide polymorphisms). This capability was highlighted in this paper for the identification of a truncated form of cystatin C in patients with multiple sclerosis and also in the discrimination between the α (15.1 kDa) and β (15.9 kDa) subunits of hemoglobin A. Quantitative comparison of post-translationally derived biomarkers or protein variants could yield valuable information about the stability or expression of the variants or post-translational modifications (PTMs) and prove to be diagnostically useful, as has been highlighted by previous groups when studying proteins present in blood.16, 76, 77
By comparing the ratio of two forms of the same antigen, the need for an internal standard is reduced. For example, α and β thalassemia are inherited diseases of red blood cells that manifests in a deficiency of the respective α and β chains in hemoglobin. The extent of the disease is dependent upon the number of loci that are affected. The SAMDI-TOF immunosensor may offer a simple method to quantitatively compare the ratio of these two subunits (without the use of an internal standard) and thus provide a prognosis for disease.
Despite the clear benefits that protein-based mass spectrometry methods offer in clinical diagnostic assays, they are still not employed in this setting. One reason for this slow acceptance stems from the usual barrier to the introduction of novel methods, but other factors include the need for technicians with specialized training and the need for methods that simplify the preparation, isolation and enrichment of the analytes.78
Standardization of analytical methods has partly been addressed by the commercial SELDI-TOF mass spectrometer. In the traditional SELDI experiment, proteins are partitioned onto a solid support through non-specific interactions with chromatographic resins arrayed onto the surface. In this way, analytes in a complex sample can be enriched through their preferential interaction with a solid phase resin, but cannot be isolated from the sample as is done with immunoassays. This lack of specificity of the plate for an analyte, and the lack of standardized pre-analytical procedures for samples give rise to peak intensity CVs of 10–40%, and limited dynamic range for protein detection in complex mixtures.79
Strategies, such as the SAMDI-TOF immunoassay, which combine well defined surface chemistries for the selective and reproducible localization of analytes with mass spectrometry may offer an alternative methodology to address many of the issues associated with standardized clinical diagnostics. Further improvements to the current assay design could be realized by the covalent attachment of the protein G (or A) to the maleimide surface through a recombinant oligo-cysteine tag.80
Such a design, combined with the known long term stability of the alkanethiolate monolayer,44, 57
could greatly extend the shelf-life of the assay as well as minimize the steps necessary to perform an immunoassay with SAMDI.