Elemental Mass Spectrometry for Absolute Intact Protein Quantification without Protein-Specific Standards: Application to Snake Venomics
Francisco Calderón‐CelisSilvia Diez-FernándezJosé M. Costa‐FernándezJorge Ruíz EncinarJuan J. CalveteAlfredo Sanz‐Medel
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Absolute protein quantification methods based on molecular mass spectrometry usually require stable isotope-labeled analogous standards for each target protein or peptide under study, which in turn must be certified using natural standards. In this work, we report a direct and accurate methodology based on capLC-ICP-QQQ and online isotope dilution analysis for the absolute and sensitive quantification of intact proteins. The combination of the postcolumn addition of 34S and a generic S-containing internal standard spiked to the sample provides full compound independent detector response and thus protein quantification without the need for specific standards. Quantitative recoveries, using a chromatographic core-shell C4 column for the various protein species assayed were obtained (96-100%). Thus, the proposed strategy enables the accurate quantification of proteins even if no specific standards are available for them. In addition, to the best of our knowledge, we obtained the lowest detection limits reported in the quantitative analysis of intact proteins by direct measurement of sulfur with ICPMS (358 fmol) and protein (ranging from 7 to 15 fmol depending on the assayed protein). The quantitative results for individual and simple mixtures of model proteins were statistically indistinguishable from the manufacturer's values. Finally, the suitability of the strategy for real sample analysis (including quantitative protein recovery from the column) was illustrated for the individual absolute quantification of the proteins and whole protein content in a venom sample. Parallel capLC-ESI-QTOF analysis was employed to identify the proteins, a prerequisite to translate the mass of quantified S for each chromatographic peak into individual protein mass.Keywords:
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A rapid, accurate, sensitive, and simple method for simultaneous speciation analysis of mercury and tin in biological samples has been developed. Integrated simultaneous sample preparation for tin and mercury species includes open focused microwave extraction and derivatization via ethylation. Capillary gas chromatography−inductively plasma mass spectrometry (CGC−ICPMS) conditions and parameters affecting the analytical performance were carefully optimized both for species-specific isotope dilution analysis of MMHg and TBT and for conventional analysis of MBT and DBT. 201Hg-enriched monomethylmercury and 117Sn-enriched tributyltin were used for species-specific isotope dilution mass spectrometry (SIDMS) analysis. As important, accurate isotope dilution analysis requires equilibration between the spike and the analyte to achieve successful analytical procedures. Since the spike stabilization and solubilization are the most critical and time-consuming steps in isotope dilution analysis, different spiking procedures were tested. Simultaneous microwave-assisted spike stabilization and solubilization can be achieved within less than 5 min. This study originally introduces a method for the simultaneous speciation and isotope dilution of mercury and tin in biological tissues. The sample throughput of the procedure was drastically reduced by fastening sample preparation and GC separation steps. The accuracy of the method was tested by both external calibration analysis and species-specific isotope dilution analysis using the first biological reference material certified for multielemental speciation (oyster tissue, CRM 710, IRMM). The results obtained demonstrate that isotope dilution analysis is a powerful method allowing the simultaneous speciation of TBT and MMHg with high precision and excellent accuracy. Analytical problems related to low recovery during sample preparation are thus minimized by SIDMS. In addition, a rapid procedure allows us to establish a performant routine method using CGC−ICPMS technique.
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Abstract In order to assess the biological function of proteins and their modifications for understanding signaling mechanisms within cells as well as specific biomarkers to disease, it is important that quantitative information be obtained under different experimental conditions. Stable isotope labeling is a powerful method for accurately determining changes in the levels of proteins and PTMs; however, isotope labeling experiments suffer from limited dynamic range resulting in signal change ratios of less than ∼20:1 using most commercial mass spectrometers. Label‐free approaches to relative quantification in proteomics such as spectral counting have gained popularity since no additional chemistries are needed. Here, we show a label‐free method for relative quantification based on the TIC from peptide MS/MS spectra collected from data‐dependent runs can be used effectively as a quantitative measure and expands the dynamic range over isotope labeling experiments allowing for abundance differences up to ∼60:1 in a screen for proteins that bind to phosphotyrosine residues.
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Quantitative data is an excellent resource in any proteomics study but is essential in many. In recent years this area has expanded from relative to absolute quantification with a wide range of methods available for absolute quantitative proteomics. In general protein quantification is based on either label-mediated or label-free strategies. Common label-mediated approaches are isotope dilution strategies, such as AQUA, coupled with mass spectrometry, where analyte signal is compared to a stable isotope labelled standard added in known abundance. These methods are suited to small-scale studies but increasing demand for large-scale proteome quantification exposed the need for alternative quantification methodologies. The QconCAT technology, first published in 2005, is a label mediated approach which utilises the principle of surrogacy to quantify analyte proteins based on a signature peptide, or peptides, for each protein. QconCATs are concatenations of quantotypic peptides for a group of proteins, the QconCAT gene is designed in silico and expressed heterologously in E.coli with [13C6]arg and [13C6]lys to elicit a stable isotope labelled multiplexed absolute quantification standard. In this thesis I describe several developments to the QconCAT production protocol. These developments reduce the production time from ~19d, using the initial method, to less than 7d. Time gains have been made across the whole workflow in the areas of protein expression, cell lysis, and product purification. Moreover verification of the QconCAT is delayed until the final product is synthesised, made possible by evidence of high quality reproducible expression. I explain how these alterations allow for production of several QconCATs in parallel, giving added efficiency. The success of the method is demonstrated through the use of multiple QconCATs. As a result of this work it is now possible to make at least eight QconCATs per week and the rate-limiting step of the quantification workflow has migrated from standard preparation to data processing. The final study in this thesis discusses methods for accurate quantification of the QconCAT protein and additional applications of QconCATs for testing mass spectrometer performance.
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Quantitative proteome analysis has become a versatile tool to understand biological functions. Although stable isotope labeling is the most reliable method for quantitative mass spectrometry, preparation of isotope-labeled compounds is time-consuming and expensive. Simple label-free approaches have been introduced, but intensity-based quantitation without standards is not generally accepted as reliable, especially for small molecules. We have developed a novel label-free quantitative proteome analysis using pseudo internal standards (PISs). This idea was derived from northern blotting analysis, in which housekeeping genes are used as standards to normalize and compare target gene expression levels in different samples. In many proteomics studies, most proteins do not change their expression levels under different conditions, and therefore, these proteins can be employed as pseudo internal standards. This new approach is simple and does not require additional standards or labeling reagents. The PIS method represents a novel approach for mass spectrometry-based comprehensive quantitatitation and may also be applicable to quantitative metabolome analysis.
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To address the challenges associated with differential expression proteomics, label-free mass spectrometric protein quantification methods have been developed as alternatives to array-based, gel-based, and stable isotope tag or label-based approaches. In this paper, we focus on the issues associated with label-free methods that rely on quantitation based on peptide ion peak area measurement. These issues include chromatographic alignment, peptide qualification for quantitation, and normalization. In addressing these issues, we present various approaches, assembled in a recently developed label-free quantitative mass spectrometry platform, that overcome these difficulties and enable comprehensive, accurate, and reproducible protein quantitation in highly complex protein mixtures from experiments with many sample groups. As examples of the utility of this approach, we present a variety of cases where the platform was applied successfully to assess differential protein expression or abundance in body fluids, in vitro nanotoxicology models, tissue proteomics in genetic knock-in mice, and cell membrane proteomics.
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In order to study the differential protein expression in complex biological samples, strategies for rapid, highly reproducible and accurate quantification are necessary. Isotope labeling and fluorescent labeling techniques have been widely used in quantitative proteomics research. However, researchers are increasingly turning to label-free shotgun proteomics techniques for faster, cleaner, and simpler results. Mass spectrometry-based label-free quantitative proteomics falls into two general categories. In the first are the measurements of changes in chromatographic ion intensity such as peptide peak areas or peak heights. The second is based on the spectral counting of identified proteins. In this paper, we will discuss the technologies of these label-free quantitative methods, statistics, available computational software, and their applications in complex proteomics studies.
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