Geoffrey Rule, Ph.D. - Principal Scientist, MilliporeSigma, Bellefonte, PA, A business of Merck KGaA, Darmstadt, Germany
Cory Muraco - Biomolecule Workflows Manager, MilliporeSigma, Bellefonte, PA, A business of Merck KGaA, Darmstadt, Germany
Use of antibodies for detection of very low analyte concentrations has been evolving since Yalow and Berson developed the radioimmunoassay in the late 1950’s1 . Despite the tremendous advancements immunoassays provided in clinical science, some immunoassays were prone to interferences from cross-reactivity and erroneous results. Later, the direct combination of immunoaffinity techniques with LC-MS would be realized as exquisitely powerful2 . The extreme selectivity of antibodies makes them indispensable when preparing complex samples. In combination with the sensitivity of MS this can lead to low limits of quantitation for important analytes.
A multitude of papers have been published on use of immunoaffinity techniques for determination not only of small molecules but for a variety of pharmaceutically and clinically relevant proteins by MS. Neubert et al.3 published a special report on the latter topics.
Reagents and materials for affinity techniques are widely available making this approach worth considering for clinical and therapeutic applications. The combination of affinity purifications, either during sample preparation or chromatography, makes a powerful technique for enrichment and analysis of low-level proteins.
Strategies for Proteins
For protein determination there are several strategies for target enrichment and analysis. For LC-MS analysis, a bottom-up approach to sample preparation is commonly used where the protein is digested enzymatically and one or more of the resulting peptides are used as surrogate markers of the protein. Immuno (affinity) enrichment is conducted either before or after the digestion or, in some cases, both. For targeting of IgG or mAbs, a Protein A or Protein G enrichment can selectivity pull out antibodies by the Fc, or tail region, from a culture broth or serum sample. Where an antibody directed towards a target protein is available, either in polyclonal or monoclonal form, the anti-analyte antibody can selectively enrich the analyte while removing undesired matrix components.
In the ideal situation, a Stable Isotope Labeled Internal Standard (SIL-IS) version of the target protein is available and added to the sample prior to the capture step. This approach may be more practical in pharma where development of SIL-IS protein can be carried out using existing methods and heavy labelled amino acids. After capture of target and SIL-IS proteins, digestion provides peptides stemming only from the target protein and SIL-IS.
An alternative is to submit the entire protein sample to digestion, adding a SIL-IS peptide either before or after this step. Antibodies toward one or more target peptides are then used to enrich these markers of the protein of interest, as well as corresponding SIL-peptides. A challenge of this approach is creating antibodies to the target peptides as this can entail time and expense. On the other hand, SIL-peptides are more readily obtained than SIL-proteins. In both cases, peptides from digestion are subjected to LC-MS(/MS) analysis for quantitation.
Affinity-based enrichment technologies
Several formats are available for affinity enrichment of target analytes including magnetic bead-based, columnbased, and in pipette tip or filter plate devices. Beads are available in a variety of types and chemistries ranging from agarose beads to silica, organic polymer, and magnetic particles. Beads are adaptable to a range of uses from individual tubes for manual preparation, or 96-well plate formats for ease in automation. High-throughput, automated sample preparation, is facilitated by robotic workstations providing either magnetic retention of beads or use of pressure to force solutions through the affinity medium.
Column-based approaches can use particle-based or monolithic structures as the affinity support, for example when using the Chromolith WP 300 Protein A column. This monolithic column features 2 µm macropores and 300 Å mesopores, enabling rapid transport of mAbs through the column without a concomitant increase in backpressure or loss of efficiency. In a simple approach to antibody quantitation, the Protein A column is used to retain mAbs from a culture broth. Unwanted culture medium components are washed away prior to mAb elution, with a low pH elution buffer, UV detection, and quantitation.
More complex methods utilize coupled-column systems of pumps, columns, and switching valves. For example, an affinity resin is prepared as a first enrichment column onto which the sample is delivered. After a period of washing, retained peptides or protein are delivered to an analytical column for separation and detection.
Chemistries to bind proteins
A variety of covalent or noncovalent attachment chemistries are available that bind proteins to solid supports. In addition to Protein A and G, noncovalent attachments are possible with other proteins (e.g., streptavidin), oligonucleotide aptamers, antibodies to a “FLAG” sequence coded into the terminal ends of recombinant proteins, and immobilized metal affinity chromatography (IMAC) resins. The latter bind and retain recombinant proteins with a histidine tail, or “His-Tag”.
For covalent attachment, the available chemistries differ with respect to the resin or solid support. Some use an epoxy group that attaches to carboxy, thiol, hydroxyl, and amine functional groups on the protein, depending on the pH of the solution. Others also exist including N-hydroxysuccinimide (NHS), and carboxy linkages.
References
- Yalow, R.S. and Berson, S.A., Immunoassay of endogenous plasma insulin in man. J Clin Inves, 1960, 39(7): 1157-1175.
- Rule, G.S. and Henion J.D., Determination of drugs from urine by on-line immunoaffinity chromatographyhigh-performance liquid chromatography-mass spectrometry. J Chrom, 1992, 582: 103-112.
- Neubert, H., et al., Protein Biomarker Quantification by Immunoaffinity Liquid Chromatography-Tandem Mass Spectrometry: Current State and Future Vision. Clin Chem, 2020, 66(2): 282-301.
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