Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.
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 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.
The authors have nothing to disclose.
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.
Name of the material | Company | Catalogue number |
---|---|---|
1000ul Deep well 96-well plates | Eppendorf | 951032786 |
Oasis HLB 1 cc cartridge plate, Sep-pak, 40 mg 96 well plate | Waters | 186003966 |
Kingfisher 96 well plate | Thermo Fisher Scientific | 97002540 |
Clear 96 well, white wall plate | Bio-Rad | HSP9601 |
Foil cover for 96 well plate | Excelscientific | 12-169 |
Axymat Sealing mat for 96 well plate | Axygen | 521-01-151 |
X pierce film | Sigma-Aldrich | Z722502 |
Kingfisher magnetic bead processor with PCR magnet head | Thermo Fisher Scientific | HSP 9601 |
Table 1: Materials
Name of the material | Company | Catalogue number | Comments (optional) |
---|---|---|---|
Urea | Sigma Aldrich | U6031 | |
Trizma base | Sigma Aldrich | T1503 | |
Dithiothreitol (DTT) | Pierce | 20291 | “no-weigh dithiothreitol” |
Iodoacetamide (IAM) | Sigma Aldrich | I1149 | |
Trypsin Gold | Promega | V5280 | |
Protein G magnetic beads, 2.8um | Invitrogen | 10004D | |
Phosphate-buffered saline (PBS) | Thermo Fisher Scientific | L5401 | |
CHAPS | Thermo Fisher Scientific | 28300 | |
Formic acid | EMD | 11670 | |
Acetonitrile | Thermo Fisher Scientific | A998-1 | |
Acetic Acid | Sigma Aldrich | 242853 |
Table 2: Reagents
Solution | Comments (optional) |
---|---|
100 mM Tris pH 8 | |
9 M urea / 30 mM DTT in 100 mM Tris (pH 8) | must be prepared fresh each time |
500 mM IAM in 100 mM Tris (pH 8) | must be prepared fresh each time |
Trypsin 1mg/mL in 100 mM Tris pH 8 | |
Trypsin inhibitor 1mg/mL in 100 mM Tris pH 8 | |
Stable isotope standard mastermix | |
Anti-peptide antibody mastermix |
Table 3: Solutions to be prepared