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There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.
The assay requires synthetic peptides and anti-peptide antibodies. Selected peptides should be unique to the protein of interest, contain between 8 and 22 amino acids, and have no known post-translational modifications. Methionine residues are generally avoided and peptides containing dibasic amino acids (e.g. KK, KR, RR) are undesirable. For this technique, it is common to use stable isotope labeled peptides as internal standards, incorporating heavy (13C and 15N) labeled amino acids at the C-terminus of the peptide (i.e. K or R labeled).
The following protocol describes an assay developed to measure the peptide GDSLAYGLR from the mouse protein Osteopontin, using anti-peptide antibodies obtained from Epitomics Inc. (Burlingame, CA) and synthetic peptides from New England Peptide (Gardner, MA). The protocol consists of three main steps (Figure 1): 1) Trypsin digestion of the complex protein mixture, 2) Enrichment of peptides 3) Analysis by mass spectrometry. It will be demonstrated on a human plasma sample spiked with the mouse Osteopontin protein.
Measured peak area ratios (light endogenous peptide relative to spiked heavy isotopically-labeled peptide) provide a quantitative measure of the target peptide. Figure 3 shows an example chromatogram of light and heavy peptides in a SISCAPA-enriched sample. Note that light and heavy peptides elute at the same time and multiple transitions can be monitored for each peptide to confirm the identity.
Figure 1. A schematic overview of the SISCAPA process. A complex protein mixture is digested into peptides. Targeted peptide analytes (endogenous analyte and a spiked stable isotope-labeled internal standard) are enriched using anti-peptide antibodies immobilized on Protein G-coated magnetic particles. Following isolation, the targeted peptides are eluted from the magnetic particles and analyzed by mass spectrometry for quantitation relative to the internal standard.
Figure 2. MS/MS spectrum of the peptide GDSLAYGLR showing selection of three fragments for SRM/MRM transitions.
Figure 3. Example chromatograms showing the peak profile of a light peptide analyte (red) and the heavy stable isotope-labeled internal standard (blue). Chromatograms for the y5 transition from the light peptide (476.3 > 579.3) and heavy stable isotope labeled standard (481.3 > 589.3) are plotted over time as they elute from the chromatography system.
The most critical step in the protocol as described is ensuring the beads remain well-mixed during the incubation period. Allowing the beads to settle to the bottom of the well/vial will result in increased variability. It is also important to spin-down any liquid that may remain on the top of the well/vial following the incubation period. Reproducible trypsin digestion is also critical. The procedure for digestion described here has been utilized extensively in our laboratory in conjunction with SISCAPA; however, it is likely alternate methods of digestion could be optimized for a given set of target proteins.15 Elution of free antibody does not interfere with detection of the peptide, likely because the antibody is excluded from the column or elutes later than the peptides, but the antibody can be crosslinked to the Protein G beads prior to affinity enrichment in large experiments or if free antibody becomes a problem. We have also found it useful to place a magnet below the sample plate on the autosampler as well as washing the trap column in the reverse direction of flow (compared to loading) to remove any residual beads or particles. In addition to the magnetic bead approach described, the technique can also be adapted to a column format (i.e. affinity chromatography).12, 16
Once the user is comfortable with the overall protocol, the technique is also amenable to several modifications that enhance the overall assay.11 First, it is possible to analyze multiple analytes in a single assay by combining antibodies in the enrichment step (i.e. multiplexing). The mass spectrometer is capable of simultaneously analyzing large numbers of analytes. Second, increasing the volume of original sample improves the sensitivity of the assay.
No conflicts of interest declared.
This work was funded by the NCI Clinical Proteomic Technology Assessment Center (CPTAC) grant (#U24 CA126476) as well as a grant from the Entertainment Industry Foundation (EIF) and the EIF Women's Cancer Research Fund to the Breast Cancer Biomarker Discovery Consortium, and generous gifts from the Keck Foundation, the Canary Foundation, and the Paul G. Allen Family Foundation.