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Stable isotope standards and capture by anti-peptide antibodies

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Stable isotope standards and capture by anti-peptide antibodies (SISCAPA) is a mass spectrometry method for measuring the amount of a protein in a biological sample.[1]

History

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Introduced in 2004, the method has been used in a variety of studies in the field of proteomics, as well as in clinical blood tests in reference laboratories, and combines the advantageous features of mass spectrometry with those of conventional immunoassays.[2] SISCAPA is used for measurement of specific pre-selected proteins and peptides (i.e., directed assays) rather than for broad exploration of sample contents (the typical objective of proteomics discovery or survey experiments).

Method

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SISCAPA is an extension of the well-known gold-standard methods of stable-isotope dilution for quantitation of small molecules by mass spectrometry (MS).[3] Rather than measure an intact protein directly by mass spectrometry, SISCAPA makes use of proteolytic digestion (e.g., with the enzyme trypsin) to cleave sample proteins into smaller peptides ideally suited to quantitation by mass spectrometry. By selecting a target peptide whose sequence occurs only in the selected target protein (a so-called “proteotypic” peptide), the target peptide can serve as a direct quantitative surrogate for the target protein (assuming the digestion process is complete, or at least reproducible). A synthetic version of the target peptide containing a stable isotope label is added in a known amount to the digested sample to serve as an internal standard (SIS). Since the target peptide and SIS are chemically indistinguishable throughout the workflow, but can be measured separately by a mass spectrometer due to the mass difference of the stable isotope label, their ratio provides the desired quantitative estimate of the target peptide amount.

The SISCAPA workflow adds a specific enrichment step to the isotope dilution method in which a selected target peptide, together with its associated SIS internal standard, is captured by a sequence-specific anti-peptide antibody. The antibody, together with the captured target peptide, is then separated from the complex sample digest, after which the highly purified peptide is eluted from the antibody and delivered to a mass spectrometer for measurement. The capture step has been implemented using antibodies bound to magnetic beads[4][5] as well as antibodies immobilized on flow-through columns.[6] Addition of this specific capture step provides two primary advantages in comparison with a conventional workflow analyzing an unfractionated sample digest: sensitivity and throughput.

The antibody can be used to capture the target peptide (and SIS) from a much larger mass of sample than could be analyzed directly by MS, thus allowing lower concentrations to be measured. In practice, assay sensitivity can be improved by 1,000-10,000-fold by this approach.

By removing the unbound (non-target) peptides present in the sample digest, the sample presented to the mass spectrometer is drastically simplified, thus reducing the need for peptide separation by liquid chromatography prior to MS analysis. In some cases liquid chromatography has been eliminated entirely, resulting in MS cycle times of 7-20 sec[7][8] rather than 5–40 minutes required in typical unfractionated digest protocols involving extensive chromatographic separation.

By virtue of the extreme specificity of mass spectrometric detection, SISCAPA assays can be combined into multiplex panels without cross-assay interference. Panels combining 22,[9] 50,[10] and 150[11] assays into a single operation have been demonstrated.

SISCAPA’s use of proteolytic digestion as a first step eliminates the protein:protein complexes (including complexes of a target protein with auto-antibodies) that cause “interferences” in protein capture assays, including conventional immunoassays (e.g., ELISA assays). Elimination of such autoantibody interferences drove the adoption[12][13] of SISCAPA as an alternative to immunoassays for clinical measurement of thyroglobulin as a marker of thyroid cancer recurrence in patients who exhibit anti-thyroglobulin autoantibodies.

A limitation of the specific peptide capture approach is the requirement for a specially-developed antibody against the selected peptide. To date several hundred anti-peptide antibody reagents have been developed for enrichment of tryptic peptides from sample digests, mainly for established clinical biomarkers (,[14]) and cancer research targets,[15] but these do not yet cover a majority of protein targets of interest in non-cancer research or clinical contexts.[16]

A variety of MS instrument types have been used for quantitation of the enriched peptides, including most frequently triple quadrupole mass spectrometers implementing the “multiple reaction monitoring” (MRM) method (a format sometimes referred to as “immuno-MRM”[17]), and MALDI-ToF (a format sometimes referred to as iMALDI[18]).

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Alternative specific enrichment methods include MSIA[19] (“mass spectrometric immunoassay”,) in which antibodies are used to enrich target proteins, which are analyzed intact by MS; and hybrid methods[20] in which antibodies are used to enrich target proteins, which are then digested prior to peptide detection by MS.

References

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  1. ^ Anderson, N. Leigh; Anderson, Norman G.; Haines, Lee R.; Hardie, Darryl B.; Olafson, Robert W.; Pearson, Terry W. (2004). "Mass Spectrometric Quantitation of Peptides and Proteins Using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA)". Journal of Proteome Research. 3 (2): 235–244. doi:10.1021/pr034086h. ISSN 1535-3893. PMID 15113099.
  2. ^ Timothy D. Veenstra (30 May 2013). Proteomic Applications in Cancer Detection and Discovery. John Wiley & Sons. pp. 263–. ISBN 978-1-118-63441-7.
  3. ^ Brun, Virginie; Masselon, Christophe; Garin, Jérôme; Dupuis, Alain (2009). "Isotope dilution strategies for absolute quantitative proteomics". Journal of Proteomics. 72 (5): 740–749. doi:10.1016/j.jprot.2009.03.007. ISSN 1874-3919. PMID 19341828.
  4. ^ Whiteaker J, Zhao L, Zhang H, Feng L, Piening B, Anderson L, et al. Antibody-based enrichment of peptides on magnetic beads for mass-spectrometry-based quantification of serum biomarkers. Anal Biochem [Internet]. 2007 Mar 1;362(1):44–54.
  5. ^ Razavi M, Leigh Anderson N, Pope ME, Yip R, Pearson TW. High precision quantification of human plasma proteins using the automated SISCAPA Immuno-MS workflow. New Biotechnology. 2016 Jan 6.
  6. ^ Ocaña MF, Neubert H. An immunoaffinity liquid chromatography-tandem mass spectrometry assay for the quantitation of matrix metalloproteinase 9 in mouse serum. Anal Biochem. 2010 Apr 15;399(2):202–10.
  7. ^ Razavi M, Frick LE, Lamarr WA, Pope ME, Miller CA, Anderson NL, et al. High-Throughput SISCAPA Quantitation of Peptides from Human Plasma Digests by Ultrafast, Liquid Chromatography-Free Mass Spectrometry. J Proteome Res. 2012 Nov 19;:121119143208008–8.
  8. ^ Razavi M, Johnson LDS, Lum JJ, Kruppa G, Anderson NL, Pearson TW. Quantification of a Proteotypic Peptide from Protein C Inhibitor by Liquid Chromatography-Free SISCAPA-MALDI Mass Spectrometry: Application to Identification of Recurrence of Prostate Cancer. Clin Chem. 2013 Oct;59(10):1514–22.
  9. ^ Razavi M, Anderson NL, Yip R, Pope ME, Pearson TW. Multiplexed longitudinal measurement of protein biomarkers in DBS using an automated SISCAPA workflow. Bioanalysis. 2016 Jul 15.
  10. ^ Whiteaker JR, Zhao L, Lin C, Yan P, Wang P, Paulovich AG. Sequential Multiplexed Analyte Quantification Using Peptide Immunoaffinity Enrichment Coupled to Mass Spectrometry. 2012 Jun 12;11(6):M111.015347–7.
  11. ^ Ippoliti PJ, Kuhn E, Mani DR, Fagbami L, Keshishian H, Burgess MW, et al. Automated Microchromatography Enables Multiplexing of Immunoaffinity Enrichment of Peptides to Greater than 150 for Targeted MS-Based Assays. Anal Chem. 2016 Aug 2;88(15):7548–55.
  12. ^ Hoofnagle AN. Serum thyroglobulin: a model of immunoassay imperfection. 2006;(8):12–4.
  13. ^ Hoofnagle AN, Roth MY. Improving the Measurement of Serum Thyroglobulin With Mass Spectrometry. J Clin Endocrinol Metab. 2013 Apr;98(4):1343–52.
  14. ^ Anderson NL. The Clinical Plasma Proteome: A Survey of Clinical Assays for Proteins in Plasma and Serum. Clin Chem. 2010 Jan 28;56(2):177–85.
  15. ^ Whiteaker JR, Zhao L, Yan P, Ivey RG, Voytovich UJ, Moore HD, et al. Peptide immunoaffinity enrichment and targeted mass spectrometry enables multiplex, quantitative pharmacodynamic studies of phospho-signaling. 2015 May 18;:mcp.O115.050351.
  16. ^ Anderson NL, Anderson NG, Pearson TW, Borchers CH, Paulovich AG, Patterson SD, et al. A human proteome detection and quantitation project. 2009 May;8(5):883–6.
  17. ^ Zhao L, Whiteaker JR, Voytovich UJ, Ivey RG, Paulovich AG. Antibody-Coupled Magnetic Beads Can Be Reused in Immuno-MRM Assays To Reduce Cost and Extend Antibody Supply. J Proteome Res. 2015 Oct 2;14(10):4425–31.
  18. ^ Jiang J, Parker CE, Fuller JR, Kawula TH, Borchers CH. An immunoaffinity tandem mass spectrometry (iMALDI) assay for detection of Francisella tularensis. Anal Chim Acta. 2007 Dec 12;605(1):70–9.
  19. ^ Nelson R, Krone J, Bieber A. Mass spectrometric immunoassay. Anal Chem. 1995
  20. ^ Ackermann BL. Hybrid immunoaffinity–mass spectrometric methods for efficient protein biomarker verification in pharmaceutical development. Bioanalysis. Future Science Ltd London, UK; 2009 May;1(2):265–8.
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