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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
A major bottleneck in high-throughput protein production is the validation step, which is why parallel and automated sample processing methods are highly desirable. Also, a miniaturized sample preparation format is preferred, as the reduction of reagent volumes significantly decreases the analysis cost per sample. We have developed an automated and miniaturized protein sequence verification protocol for recombinant proteins utilizing peptide mass fingerprinting and MS/MS analysis. The integrated selective enrichment target (ISET) platform, previously developed in our group, with its dual functionality, being both a sample preparation platform and a MALDI target plate, is employed. All steps including immobilized metal ion affinity chromatography of protein on cobalt-loaded beads, tryptic digestion, and MALDI MS analysis are performed in an array format, without any sample transfers, on the same ISET chip. The automated configuration reduced the sample preparation time significantly. Starting with crude lysate, a full plate of 48 purified, digested samples prepared for MALDI-MS can be generated in 4 h, with only 30 min of operator involvement. This paper demonstrates the utility of the method by parallel analysis of 45 His-tagged human recombinant proteins.
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