Top-down proteomics analysis
As a tool for proteins and peptides separated by electrophoresis or chromatography, and with or without previous fragmentation, mass spectrometry has been applied in several different ways; the two approaches are top-down and bottom-up MS. Top-down MS is the direct analysis of proteins on the intact level. Bottom-up approach identifies proteins following enzymatic or chemical digestion of the sample resulting in the formation of much smaller peptide fragments. These smaller protein segments are much easier to analyze with low resolution MS instruments and therefore bottom-up peptide analysis is currently the most popular MS-based proteomics approach (Han, Jin et al. 2006).
The strength of top-down proteomics approach lies in the direct detection of the native molecular mass of biological protein species. Mass spectrometry detection provides information for the native protein, and also for the natively occurring small peptides, biologically generated protein cleavages, post-translational modifications or point mutations – all of which are postulated to be relevant in many diseases and other biological processes occurring in cells. Other major advantage of top-down approach is the simplified sample preparation that does not necessitate enzymatic or chemical digestion prior to analysis. Scientist using top-down approach in studying proteins are mainly oriented towards addressing clinical questions – using population screening in the complicated process of identification and validation of potential biomarkers (Whitelegge, Halgand et al. 2006).
Several techniques are favored in top-down proteomics research platform. SELDI-TOF MS is a widely used biomarker discovery method that combines the selectivity of chromatography and sensitivity of mass spectrometry detection. The major challenge for this approach is the requirement for off-line enrichment and purification of the selected biomarker candidates, followed by MS/MS identification using different MS platform(Reid and McLuckey 2002).
MALDI-TOF MS has also found its way in implementing in top-down proteomics quest for novel biomarkers. One of the disadvantages of the methods used in routine clinical biomarker discovery techniques is the complicated sample preparation which requires chromatographic or electrophoretic separation of the targeted protein. Immunoaffinity capture of a protein directly from a biological sample is a base method used in all immunoassays (such as ELISA). Therefore, by combining the selectivity of immunoaffinity capture and the specificity of mass spectrometry, a novel methodology has been developed in recent years- Mass Spectrometry Immunoassay (MSIA).
In our work we have developed several assays using MSIA approach. Using this novel platform we were able to analyze several proteins, some of which have been introduced as potential biomarkers for several neurological diseases. An introduction to the overall procedure and application of this technique will be introduced through the development of Cystatin C MSIA assay.
Mass spectrometry immunoassay (MSIA)
Mass Spectrometry Immunoassay (MSIA) is a novel approach that has been employed for both, qualitative and quantitative characterization of body fluid proteins. From the methodology point of view, it is a combination of today’s predominant technology involved in routine clinical practice – immunoassay for targeted protein affinity extraction assessment and uses mass spectrometry detection (MALDI-TOF-MS) for achieving the specificity necessary for this type of analyses. This approach lacks the disadvantage demonstrated by the immunoassays – inability to detect protein variants, post translational modifications or mutations. On the contrary, MSIA gives onset into the detailed intrinsic protein characteristics, both for high or low abandoned proteins. One great advantage of this hybrid methodology is the simplicity of protein extraction from complicated biological samples. The basic concept of the analysis includes a two-step approach; first, proteins are captured by a principle of microscale affinity by aspiration/ dispense cycles on an antibody-derivatized affinity pipette, and in the second step eluted proteins are subsequently analyzed using MALDI-TOF-MS (Fig.4)
Fig. 4. Mass spectrometry immunoassay scheme
In the initial stage of this method’s development, agarose beads derivatized with an affinity ligand (e.g. polyclonal antibodies) were used to create a μL-scale column inside a micropipette tip (Krone, Nelson et al. 1996). These columns are further derivatized with a corresponding antibody toward the targeted protein. Additional activation of the beads is necessary prior to the derivatization process. A summary of the consecutive steps in activation and derivatization of the affinity pipettes is presented in Figure 5.
Fig. 5. Affinity pipettes activation and derivatization procedure
When the affinity pipettes are coated with the corresponding antibody, further activation of the surface is needed in order to favor protein binding. Using Multimek 96 automated 96-channel pipettor (Beckman Coulter, Brea, CA, USA), the antibody coated affinity pipettes were rinsed with buffer (PBS, with 0,1%TWEEN) in 10 aspiration/dispense cycles. Next, pipettes were immersed into a microplate containing the sample (serum, plasma, urine or CSF). Following additional buffer rinse (in order to elute the non-specific bounded proteins or other constitutes origin from the sample) and two water rinse cycles, retrieved targeted protein on the affinity pipette is ready for discharge. Figure 6 illustrates the steps in this analysis.
Fig. 6. Steps in MSIA protein analysis
Preparing for the second step of detection - the mass spectrometry detection, captured protein is eluted directly on the MALDI target, by 6 μL aliquots of MALDI matrix (α-cyano-4-hydroxycinnamic acid in aqueous solution containing 33% (v/v) acetonitrile and 0.4% (v/ v) trifluoroacetic acid). Using this acidic matrix, captured protein are dissociated from the antibodies and eventually dispensed onto the target. Following drying and visual inspection of the sample spots, linear mass spectra were acquired using delayed extraction mode with 1.7 kV draw out pulse, 200 ns delay and a full accelerating potential of 20 kV.
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MSIA |
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Qualitative MSIA |
Quantitative MSIA |
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Primary goal |
Population proteomic studies |
Biomarker discovery and validation |
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Samples |
Large cohort of samples |
Control and disease samples spiked with reference protein |
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Assay development |
Affinity pipettes immobilized with antibody toward targeted protein |
Affinity pipettes immobilized with two antibodies – toward targeted protein and IRS |
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Immunoaffinity separation |
Retrieval of targeted protein |
Retrieval of targeted protein and the IRS |
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Mass spectrometry detection |
MALDI-TOF MS |
MALDI-TOF MS |
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Results |
Detection of protein variants, post-translational modification and point mutation |
Calculating concentration of native protein and protein variants |
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Additional analyses |
Enzymatic digestion |
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Table 2. Differences between qualitative and quantitative MSIA approaches
Although introduced in the recent years, this technique has already been implemented in developing qualitative and quantitative assays for several protein and protein variants using different biological samples as medium. There is one basic difference regarding these two approaches (Table 2). When using MSIA platform for qualitative analysis, affinity pipettes are derivatized only with antibody towards the targeted protein; additional antibody must be fitted into the affinity micocolumn in order to retrieve another protein in the quantitative assay. In quantitative MSIA, affinity pipettes are derivatized with a secondary antibody, toward a protein termed as internal reference standard (IRS). Choosing the IRS is one of the critical steps in developing of the assay, due to the high criteria required for such a protein. An important prerequisite for an IRS is that it should not be present in human plasma or serum (or other biological fluids, as well), so that its spiked concentration in the analytical samples is always constant. Also, the signal on the mass spectrometer produced by the IRS should be in close proximity to the signal of the targeted protein, in order to be able to use the same MS acquisition parameters for both proteins. The goal in developing qualitative assays is determination and identification of existing or novel protein variants and point mutations. In these type of analyses, a large cohort of samples is required for analyses in order to delineate between the "wild" isoforms (post-translational modifications, or additional derivatization) present in majority of samples, and which subsequently are termed as "normal" and the pathological variants which are only present in a small number of samples or in samples from patients with certain diseases. In quantitative assays we are able to calculate the exact concentration of each variant, which, again, by screening populations, can provide information about the range of "normal" concentration distribution of the variant and the protein in general. These correlations can further be used in discussing the potential biomarker capacity of a certain protein or variant. Presented here are results of the developed MSIA qualitative and quantitative assays for determination of cystatin C and its variants.
