Immunoproteomics Using Polyclonal Antibodies and Stable Isotope–labeled Affinity-purified Recombinant Proteins

Abstract

The combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope-labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A-coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope-labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng Mass spectrometry-based proteomics is fast developing in the direction of clinical applications. Therefore, reliable quantification methods for absolute protein concentration determination are indispensible tools for future applications. So far, enzyme-linked immunosorbent assays and similar antibodybased methods excel in the sensitive detection of low levels of proteins in complex matrices, whereas mass spectrometry enables unbiased approaches and can provide unsurpassed specificity. The fact that most proteomes have a very high dynamic range between high and low abundant proteins, in particular for clinical samples, such as plasma and serum, often makes it necessary to use protein depletion of the most abundant proteins (1, 2) and/or elaborate fractionations (3-5) before running the mass spectrometry analysis. This has prompted several investigators to introduce a protein or peptide capture step using specific antibodies to allow for immunoaffinity enrichment prior to the MS analysis. In this way, a "sandwich" assay is obtained, but instead of having a readout in the analysis step based on a second antibody, the analysis step is performed using MS. In such an approach, either the intact protein is captured using an anti-protein antibody (6) or a peptide derived from the protein is captured using an antipeptide antibody that has been raised to the target peptide of interest (7)(8)(9)(10)(11). This is the principle behind stable isotope standards and capture by anti-peptide antibodies (SISCAPA), 1 developed by

Targeted Proteomics-Driven Computational Modeling of Macrophage S1P Chemosensing

“…S3). Correlation between transcript and protein absolute abundance values has been reported previously for Escherichia coli (65,66), Saccharomyces cerevisiae (65,(67)(68)(69)(70), Schizosaccharomyces pombe (39), the HeLa (human epithelial) cell line (71,72), the NIH/3T3 (mouse fibroblast) cell line (73), and for 12 human tissues (74). These correlations are consistent with the observation that transcript abundance is the major determinant of protein abundance in mouse dendritic cells (75).…”

Quantitative and Selective Analysis of Feline Growth Related Proteins Using Parallel Reaction Monitoring High Resolution Mass Spectrometry

“…In this study, a recently launched product named QPrEST™ was used as internal standard. QPrESTs™ are 50–150 amino acid long sequence segments of human proteins with heavy isotope-labelled ( 15 N, 13 C) Lysine and Arginine, developed within the Human Protein Atlas project [15–17]. QPrESTs™ are digested together with the sample giving the potential advantage that incomplete or unspecific digestion does not corrupt the results, which can be an issue using synthetic isotope-labelled peptides.…”

“…There are variants of QconCAT available, including reagents with natural flanking sequences [20]. QPrESTs™ are produced from an already existing library of well validated constructs, covering more than 80% of the human protein-coding genes [17]. …”

Commercially available antibodies can be applied in quantitative multiplexed peptide immunoaffinity enrichment targeted mass spectrometry assays

“…Based on the results, we estimate an overall success rate in developing working assays of ∼14% (combining success rates in detection and qualification of assays in MRM). This success rate is lower than a previous investigation using polyclonal antibodies generated against protein fragments for peptide immunoaffinity enrichment , possibly because use of polyclonals can increase the likelihood of a working antibody by enriching multiple peptides (i.e., multiple epitopes) for each protein target. Limitations of polyclonal antibodies (e.g., limited resource, batch‐to‐batch variation) make the use of mAbs more attractive for a distributable and standardizable assay reagent.…”