A method (denoted SISCAPA) for quantitation of peptides in complex digests is described. In the method, anti-peptide antibodies immobilized on 100 nanoliter nanoaffinity columns are used to enrich specific peptides along with spiked stable-isotope-labeled internal standards of the same sequence. Upon elution from the anti-peptide antibody supports, electrospray mass spectrometry is used to quantitate the peptides (natural and labeled). In a series of pilot experiments, tryptic test peptides were chosen for four proteins of human plasma (hemopexin, alpha1 antichymotrypsin, interleukin-6, and tumor necrosis factor-alpha) from a pool of 10,203 in silico tryptic peptide candidates representing 237 known plasma components. Rabbit polyclonal antibodies raised against the chosen peptide sequences were affinity purified and covalently immobilized on POROS supports. Binding and elution from these supports was shown to provide an average 120-fold enrichment of the antigen peptide relative to others, as measured by selected ion monitoring (SIM) or selected reaction monitoring (SRM) electrospray mass spectrometry. The columns could be recycled with little loss in binding capacity, and generated peptide ion current measurements with cycle-to-cycle coefficients of variation near 5%. Anti-peptide antibody enrichment will contribute to increased sensitivity of MS-based assays, particularly for lower abundance proteins in plasma, and may ultimately allow substitution of a rapid bind/elute process for the time-consuming reverse phase separation now used as a prelude to online MS peptide assays. The method appears suitable for rapid generation of assays for defined proteins, and should find application in the validation of diagnostic protein panels in large sample sets.
Abstract. In the mammalian host, the unicellular flagellate Trypanosoma brucei is covered by a dense surface coat that consists of a single species of macromolecule, the membrane form of the variant surface glycoprotein (mfVSG). After uptake by the insect vector, the tsetse fly, bloodstream-form trypanosomes differentiate to procyclic forms in the fly midgut.
The inability to quantify large numbers of proteins in tissues and biofluids with high precision, sensitivity, and throughput is a major bottleneck in biomarker studies. We previously demonstrated that coupling immunoaffinity enrichment using anti-peptide antibodies (SISCAPA) to multiple reaction monitoring mass spectrometry (MRM-MS) produces Immunoprecipitation MRM-MS (immuno-MRM-MS) assays that can be multiplexed to quantify proteins in plasma with high sensitivity, specificity, and precision. Here we report the first systematic evaluation of the interlaboratory performance of multiplexed (8-plex) immuno-MRM-MS in three independent labs. A staged study was carried out in which the effect of each processing and analysis step on assay coefficient of variance, limit of detection, limit of quantification, and recovery was evaluated. Limits of detection were at or below 1 ng/ml for the assayed proteins in 30 l of plasma. Assay reproducibility was acceptable for verification studies, with median intra-and interlaboratory coefficients of variance above the limit of quantification of 11% and <14%, respectively, for the entire immuno-MRM-MS assay process, including enzymatic digestion of plasma. Trypsin digestion and its requisite sample handling contributed the most to assay variability and reduced the recovery of target peptides from digested proteins. Using a stable isotope-labeled protein as an internal standard instead of stable isotope-labeled peptides to account for losses in the digestion process nearly doubled assay accuracy for this while improving assay precision 5%. Our results demonstrate that multiplexed immuno-
The lack of sensitive, specific, multiplexable assays for most human proteins is the major technical barrier impeding development of candidate biomarkers into clinically useful tests. Recent progress in mass spectrometrybased assays for proteotypic peptides, particularly those with specific affinity peptide enrichment, offers a systematic and economical path to comprehensive quantitative coverage of the human proteome. A complete suite of assays, e.g. two peptides from the protein product of each of the ϳ20,500 human genes (here termed the human Proteome Detection and Quantitation project), would enable rapid and systematic verification of candidate biomarkers and lay a quantitative foundation for subsequent efforts to define the larger universe of splice variants, post-translational modifications, protein-protein interactions, and tissue localization.
The differentiation of mammalian stage Trypanosoma brucei bloodstream forms comprising predominantly parasites of intermediate and stumpy morphology to the procyclic forms characteristic for the insect midgut stage was studied in vitro. Differentiation of the cell population occurred synchronously as judged by the synthesis of the surface glycoprotein, procyclin, characteristic of the arising procyclic forms and the loss of the membraneform variant surface glycoprotein, the coat protein of bloodstream forms. The change in surface antigens took place within 12 h in the absence of cell growth; subsequently, the procyclic cells divided exponentially. As defined in this study, T. brucei may be a useful model to follow other changes in gene expression, metabolism or ultrastructure during differentiation of a unicellular eucaryote.The discovery of several peculiarities have promoted the unicellular parasitic protozoan, Trypanosoma brucei, to a model organism in biochemistry and molecular biology. The unusual features range from the organization of the nuclear and mitochondrial genome, the processing of pre-mRNAs by trans-splicing and the unique editing in the formation of mitochondrial RNA to the compartmentalization of the glycolytic enzymes in the glycosome and the structure of the cell surface [l -61. Moreover, because trypanosomes alternate between a mammal and an insect (the tsetse fly) as hosts, they must adjust their lifestyle to drastic changes in environment and, therefore, lend themselves to studies on differentiation between successive stages in the life cycle.
Stable isotope standards and capture by antipeptide antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution and detection by multiple reaction monitoring mass spectrometry to provide quantitative measurement of peptides as surrogates for their respective proteins. In this report, we describe a feasibility study to determine the success rate for production of suitable antibodies for SISCAPA assays in order to inform strategies for large-scale assay development. A workflow was designed that included a multiplex immunization strategy in which up to five proteotypic peptides from a single protein target were used to immunize individual rabbits. A total of 403 proteotypic tryptic peptides representing 89 protein targets were used as immunogens. Antipeptide antibody titers were measured by ELISA and 220 antipeptide antibodies representing 89 proteins were chosen for affinity purification. These antibodies were characterized with respect to their performance in SISCAPA-multiple reaction monitoring assays using trypsin-digested human plasma matrix. More than half of the assays generated were capable of detecting the target peptide at concentrations of less than 0.5 fmol/l in human plasma, corresponding to protein concentrations of less than 100 ng/ml. The strategy of multiplexing five peptide immunogens was successful in generating a working assay for 100% of the targeted proteins in this evaluation study. These results indicate it is feasible for a single laboratory to develop hundreds of assays per year and allow planning for cost-effective generation of SISCAPA assays. Molecular & Cellular Proteomics 10: 10.1074/ mcp.M110.005645, 1-10, 2011.Highly specific and sensitive assays (e.g. immunoassays) are not available for quantifying the vast majority of human proteins, and de novo assay generation is associated with a high cost and long lead time. Consequently, although genomic and proteomic technologies are used to routinely identify many hundreds of candidate biomarkers for a given disease, very few undergo further verification and validation, which require a quantitative assay. This conundrum is likely a major contributing factor to the highly inefficient translation of candidate biomarkers into clinical use (1-3).Multiple reaction monitoring mass spectrometry (MRM-MS) 1 has been used for decades in clinical reference laboratories for accurate quantitation of small molecules in plasma, such as drug metabolites or metabolites that accumulate as a result of inborn errors of metabolism (4, 5). More recently, MRM-MS has been adapted to measure the concentrations of candidate protein biomarkers in plasma and cell lysates (6 -11). 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 measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, couple...
A SISCAPA (stable isotope standards and capture by antipeptide antibodies) method for specific antibody-based capture of individual tryptic peptides from a digest of whole human plasma was developed using a simplified magnetic bead protocol and a novel rotary magnetic bead trap device. MS is the method of choice for identification of peptides in digests of biological samples based on the power of MS to detect the chemically well defined masses of both peptides and their fragments produced by processes such as CID. This high level of structural specificity is also critical in improving peptide (and protein) quantitation because it overcomes the well known problems inherent in classical immunoassays related to limited antibody specificity, dynamic range, and multiplexability. In principle, a quantitative peptide assay using MRM 1 detection in a triple quadrupole mass spectrometer should have nearly absolute structural specificity, a dynamic range of ϳ1eϩ4, and the ability to multiplex measurements of hundreds of peptides per sample (1). These properties suggest that MS-based methods could ultimately replace classical immunoassay technologies in many research and clinical applications.An important limitation of present peptide MRM measurements is sensitivity. The most sensitive widely used quantitative MS platforms use nanoflow chromatography and ESI to deliver trace amounts of peptides to the mass spectrometer. However, these processes are limited in the total amount of peptide that can be applied while retaining maximum sensitivity (typically limited to ϳ1 g of total peptide sample, i.e. the product obtained from digesting ϳ14 nl of plasma). The lower cutoff for detecting proteins in a digest of unfractionated plasma by this approach appears to be in the neighborhood of 1-20 g/ml plasma concentration, which would restrict analysis to the top 100 or so proteins in plasma (1).The sensitivity of MS assays can be substantially increased by fractionating the sample at the level of intact proteins, the tryptic peptides derived from them, or both. For example, immunodepletion of the six most abundant plasma proteins, removes ϳ85% of the protein mass (2) and results in an increase of ϳ7-fold in the signal-to-noise of MRM measurements of peptides from the remaining proteins after digestion (1). Similarly chromatographic fractionation by strong cation exchange provides another major improvement in sensitivity (3). However, increased sample fractionation brings with it the disadvantages of increased cost and time, the risk of losing specific components, and the continued requirement for very high resolution (lengthy, low throughput) reversed phase nanoflow chromatography en route to the ESI source.An alternative fractionation approach, used in the SISCAPA method, enriches specific target peptides through capture by anti-peptide antibodies, thus circumventing these disadvantages for preselected targets (4). In its initial implementation, SISCAPA used very small (ϳ10-nl) columns of POROS chro-
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