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Inflammatory responses and tumor developments are closely related, with interleukin-6 (IL-6) playing important roles in both processes. IL-6 has been extensively identified as a potential tumor biomarker. This study developed an isotope dilution mass spectrometry (IDMS) method for quantifying IL-6 based on signature peptides. These peptides were screened by excluding those with missed cleavage or post-translational modification. The method's accuracy was verified using amino acid-based IDMS, in which purified IL-6 protein samples were quantified after hydrolyzing them into amino acids, and no significant difference was observed (p-value < 0.05). The method demonstrated good linearity and sensitivity upon testing. The specificity and matrix effect of the method were verified, and a precision study showed that the coefficient of variation was less than 5% for both the intra-day and inter-day tests. Compared to immunoassays, this method offers distinct advantages, such as the facilitation of multi-target analysis. Furthermore, the peptides used in this study are much more convenient for storage and operation than the antibodies or purified proteins typically used in immunoassays. [ABSTRACT FROM AUTHOR]

This study was conducted to optimize a targeted plant proteomics approach from signature peptide selection and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analytical method development and optimization to sample preparation method optimization. Three typical protein extraction and precipitation methods, including trichloroacetic acid (TCA)/acetone method, phenol method, and TCA/acetone/phenol method, and two digestion methods, including trypsin digestion and LysC/trypsin digestion, were evaluated for selected proteins related to the impact of engineered nanomaterials (ENMs) on wheat (Triticum aestivum) plant growth. In addition, we compared two plant tissue homogenization methods: grinding freeze-dried tissue and fresh tissue into a fine powder using a mortar and pestle aided with liquid nitrogen. Wheat plants were grown under a 16 h photoperiod (light intensity 150 μmol·m-2·s-1) for 4 weeks at 22 °C with a relative humidity of 60% and were watered daily to maintain a 70-90% water content in the soil. Processed samples were analyzed with an optimized LC-MS/MS method. The concentration of selected signature peptides for the wheat proteins of interest indicated that the phenol extraction method using fresh plant tissue, coupled with trypsin digestion, was the best sample preparation method for the targeted proteomics study. Overall, the optimized approach yielded the highest total peptide concentration (68,831 ng/g, 2.4 times the lowest concentration) as well as higher signature peptide concentrations for most peptides (19 out of 28). In addition, three of the signature peptides could only be detected using the optimized approach. This study provides a workflow for optimizing targeted proteomics studies.

Inflammatory responses and tumor developments are closely related, with interleukin-6 (IL-6) playing important roles in both processes. IL-6 has been extensively identified as a potential tumor biomarker. This study developed an isotope dilution mass spectrometry (IDMS) method for quantifying IL-6 based on signature peptides. These peptides were screened by excluding those with missed cleavage or post-translational modification. The method’s accuracy was verified using amino acid-based IDMS, in which purified IL-6 protein samples were quantified after hydrolyzing them into amino acids, and no significant difference was observed (p-value < 0.05). The method demonstrated good linearity and sensitivity upon testing. The specificity and matrix effect of the method were verified, and a precision study showed that the coefficient of variation was less than 5% for both the intra-day and inter-day tests. Compared to immunoassays, this method offers distinct advantages, such as the facilitation of multi-target analysis. Furthermore, the peptides used in this study are much more convenient for storage and operation than the antibodies or purified proteins typically used in immunoassays.

Milk is one of the most common and widespread allergenic foods. Milk allergy is an adverse immunological reaction to milk proteins of different mammalian species. β-Lactoglobulin (β-LG) is one of the main allergenic proteins of cow's milk. There is an urgent need to develop an accurate and traceable method to accurately quantify β-LG. Based on the known β-lactoglobulin sequence, the Skyline tool was used to simulate the β-lactoglobulin digestion process. After digestion of β-LG with trypsin, the tryptic peptides in the samples were detected selectively by MS/MS. In short, the peptides were searched with MS full scan of respective digested milk protein. Three characteristic peptides of β-lactoglobulin were screened by primary local sequence search tool (basic local alignment search tool, BLAST) and Uniprot database. Finally, one of the characteristic peptides with the highest response intensity and the best stability was selected for further verification by HPLC-MS/MS and quantitative studied by multiple reaction monitoring (MRM). In this work, the specific peptides were quantified by isotope dilution mass spectrometry (IDMS) after trypsin digestion of β-LG. At the same time, the effects of deuterium-labeled characteristic peptide IDAL*NENK(D6-Leu) and carbon-nitrogen dual-labeled characteristic peptide IDAL*NENK(13C6,15N-Leu) as internal standards on chromatographic behavior and detection results were investigated, and the methodology was verified. The method showed a good linear relationship within its own range. The limits of detection and limits of quantification were 0.001 9-0.002 2 g/L and 0.006 4-0.007 4 g/L, respectively. The recoveries ranged from 90.1% to 102.7%, and the coefficients of variation (CVs) were less than 8.0%. Analysis of 5 samples from the domestic market showed CVs were less than 6.5%. Deuterium-labeled peptides with lower synthetic cost could be used as reliable substitutes for 13C and 15N-labeled peptides in protein quantification. The established quantitative method of isotope dilution mass spectrometry had strong anti-interference ability, high sensitivity, high accuracy and good reproducibility. It is expected that the comparability of β-LG quantitative results between different laboratories will be improved. In addition, the combination of new ultrasensitive mass spectrometry with stable-isotope labelling techniques has advanced to a point that some studies with radioactive isotopes can now be readily replaced by stable-isotope techniques. This trend is expected to continue and this isotope method will be a key component of the design of new clinical studies currently under way. At the same time, with the widespread use of stable isotope markers in food allergy, metabolomics, proteomics, clinical pharmacology and other fields, the demand for stable isotope labeled compounds is expected to further increase.

The aim of this work was to develop a liquid chromatography-tandem mass spectrometry method for the determination of milk allergen and egg allergen in food products. Signature peptides GGLEPINFQTAADQAR, VGINYWLAHK, VLVLDTDYK, FFVAPFPEVFGK, and NAVPITPTLNR were confirmed and synthesized as the quantitative peptide of ovalbumin, α-lactalbumin, β-lactoglobulin, αS1-casein and αS2-casein, the relative isotope-labeled internal standards were used in the quantitative analysis. Linear range was in the range of 0.5–5000.0 nmol/L for egg and milk allergen in bread, cake, cookie, rice crust and wheat flour samples with free from egg and milk, the limits of detection of milk allergens and egg allergen were in the range between 0.94 mg/100 g and 56.71 mg/100 g, limits of quantification of milk allergens and egg allergen were in the range between 2.36 mg/100 g and 141.78 mg/100 g. The recoveries ranged from 76.7 % to 122.8 %, the relative standard deviations were in the range of 1.60 %–15.60 %. The developed method has been successfully used for the detection of egg and milk allergen in various food samples.

Milk is one of the most common and widespread allergenic foods. Milk allergy is an adverse immunological reaction to milk proteins of different mammalian species. β-Lactoglobulin (β-LG) is one of the main allergenic proteins of cow's milk. There is an urgent need to develop an accurate and traceable method to accurately quantify β-LG. Based on the known β-lactoglobulin sequence, the Skyline tool was used to simulate the β-lactoglobulin digestion process. After digestion of β-LG with trypsin, the tryptic peptides in the samples were detected selectively by MS/MS. In short, the peptides were searched with MS full scan of respective digested milk protein. Three characteristic peptides of β-lactoglobulin were screened by primary local sequence search tool (basic local alignment search tool, BLAST) and Uniprot database. Finally, one of the characteristic peptides with the highest response intensity and the best stability was selected for further verification by HPLC-MS/MS and quantitative studied by multiple reaction monitoring (MRM). In this work, the specific peptides were quantified by isotope dilution mass spectrometry (IDMS) after trypsin digestion of β-LG. At the same time, the effects of deuterium-labeled characteristic peptide IDAL*NENK(D6-Leu) and carbon-nitrogen dual-labeled characteristic peptide IDAL*NENK(13C6,15N-Leu) as internal standards on chromatographic behavior and detection results were investigated, and the methodology was verified. The method showed a good linear relationship within its own range. The limits of detection and limits of quantification were 0.001 9-0.002 2 g/L and 0.006 4-0.007 4 g/L, respectively. The recoveries ranged from 90.1% to 102.7%, and the coefficients of variation (CVs) were less than 8.0%. Analysis of 5 samples from the domestic market showed CVs were less than 6.5%. Deuterium-labeled peptides with lower synthetic cost could be used as reliable substitutes for 13C and 15N-labeled peptides in protein quantification. The established quantitative method of isotope dilution mass spectrometry had strong anti-interference ability, high sensitivity, high accuracy and good reproducibility. It is expected that the comparability of β-LG quantitative results between different laboratories will be improved. In addition, the combination of new ultrasensitive mass spectrometry with stable-isotope labelling techniques has advanced to a point that some studies with radioactive isotopes can now be readily replaced by stable-isotope techniques. This trend is expected to continue and this isotope method will be a key component of the design of new clinical studies currently under way. At the same time, with the widespread use of stable isotope markers in food allergy, metabolomics, proteomics, clinical pharmacology and other fields, the demand for stable isotope labeled compounds is expected to further increase. [ABSTRACT FROM AUTHOR]

Natural peptides (NPs) are ubiquitous throughout the organism with diversified structures and wide dynamic concentration range, which leads to serious challenge for their comprehensive analysis. Herein, a pseudotargeted peptidomics strategy based on dynamic multiple reaction monitoring (DMRM) with high-throughput data acquisition and accurate quantification was established. Specifically, an in-house software Pep-MRMer was developed for extraction of ions information of peptides, followed by de-redundancy and fusion to construct a transition list. Additionally, retention time (RT) calibration method was optimized by using high content components for effective transference of ion pairs between different detection conditions (e.g., instruments, chromatographic gradients). This strategy was applied for profiling NPs and screening signature peptides for species authentication of Pheretima, a multi-source animal-derived drug widely used in Asian countries. In all, a total of 3307 peptides had excellent quantitative detection after transference. Subsequently, nine signature peptides with extraordinary specificity were screened, identified, and confirmed by comparing with the synthetic standards. Ultimately, a 10 min specific chromatogram based on signature peptides was developed, which realized rapid and accurate identification of Pheretima. Taken together, this research provides a robust strategy for species discrimination of animal-derived drugs without adequate quality control measures.

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Staphylococcal food poisoning outbreaks are caused by the ingestion of food contaminated with staphylococcal enterotoxins (SEs). Among the 27 SEs described in the literature to date, only a few can be detected using immuno-enzymatic-based methods that are strongly dependent on the availability of antibodies. Liquid chromatography, coupled to high-resolution mass spectrometry (LC-HRMS), has, therefore, been put forward as a relevant complementary method, but only for the detection of a limited number of enterotoxins. In this work, LC-HRMS was developed for the detection and quantification of 24 SEs. A database of 93 specific signature peptides and LC-HRMS parameters was optimized using sequences from 24 SEs, including their 162 variants. A label-free quantification protocol was established to overcome the absence of calibration standards. The LC-HRMS method showed high performance in terms of specificity, sensitivity, and accuracy when applied to 49 enterotoxin-producing strains. SE concentrations measured depended on both SE type and the coagulase-positive staphylococci (CPS) strain. This study indicates that LC-MS is a relevant alternative and complementary tool to ELISA methods. The advantages of LC-MS clearly lie in both the multiplex analysis of a large number of SEs, and the automated analysis of a high number of samples. [ABSTRACT FROM AUTHOR]

Staphylococcal food poisoning outbreaks are caused by the ingestion of food contaminated with staphylococcal enterotoxins (SEs). Among the 27 SEs described in the literature to date, only a few can be detected using immuno-enzymatic-based methods that are strongly dependent on the availability of antibodies. Liquid chromatography, coupled to high-resolution mass spectrometry (LC-HRMS), has, therefore, been put forward as a relevant complementary method, but only for the detection of a limited number of enterotoxins. In this work, LC-HRMS was developed for the detection and quantification of 24 SEs. A database of 93 specific signature peptides and LC-HRMS parameters was optimized using sequences from 24 SEs, including their 162 variants. A label-free quantification protocol was established to overcome the absence of calibration standards. The LC-HRMS method showed high performance in terms of specificity, sensitivity, and accuracy when applied to 49 enterotoxin-producing strains. SE concentrations measured depended on both SE type and the coagulase-positive staphylococci (CPS) strain. This study indicates that LC-MS is a relevant alternative and complementary tool to ELISA methods. The advantages of LC-MS clearly lie in both the multiplex analysis of a large number of SEs, and the automated analysis of a high number of samples.

The use of multiple signature peptides for the quantification of proteins by digestion and LC-MS/MS is reviewed and evaluated here. A distinction is made based on the purpose of the use of multiple peptides: confirmation of the protein concentration, discrimination between different protein forms or species and in vivo biotransformation. Most reports that describe methods with at least two peptides use these for confirmation, but it is not always mentioned how the peptides are used and how possible differences in concentration between the peptides are handled. Differences in concentration are often reported in the case of monitoring different protein forms or in vivo biotransformation, and this offers insight into the biological fate of the protein.

Natural peptides (NPs) are ubiquitous throughout the organism with diversified structures and wide dynamic concentration range, which leads to serious challenge for their comprehensive analysis. Herein, a pseudotargeted peptidomics strategy based on dynamic multiple reaction monitoring (DMRM) with high-throughput data acquisition and accurate quantification was established. Specifically, an in-house software Pep-MRMer was developed for extraction of ions information of peptides, followed by de-redundancy and fusion to construct a transition list. Additionally, retention time (RT) calibration method was optimized by using high content components for effective transference of ion pairs between different detection conditions (e.g., instruments, chromatographic gradients). This strategy was applied for profiling NPs and screening signature peptides for species authentication of Pheretima, a multi-source animal-derived drug widely used in Asian countries. In all, a total of 3307 peptides had excellent quantitative detection after transference. Subsequently, nine signature peptides with extraordinary specificity were screened, identified, and confirmed by comparing with the synthetic standards. Ultimately, a 10 min specific chromatogram based on signature peptides was developed, which realized rapid and accurate identification of Pheretima. Taken together, this research provides a robust strategy for species discrimination of animal-derived drugs without adequate quality control measures. [ABSTRACT FROM AUTHOR]

Fraud in milk and dairy products occurs when cow milk is added to sheep and goat milk for economic reasons. No reliable, selective, and sensitive method exists for quantifying the milk percentage of different species. This work reports the development and validation of a proteomics-based method for the qualitative detection and quantitative determination of cow, sheep, and goat milks in the raw materials used for dairy products. β-Lactoglobulin was selected as the protein marker because it is a major protein in milk and whey powder. The tryptic peptides LSFNPTQLEEQCHI and LAFNPTQLEGQCHV were used as signature peptides for cow milk and for sheep and goat milks, respectively. The winged peptides LKALPMHIRLSFNPTQL*EEQCHI* and LKALPMHIRLAFNPTQL*EGQCHV* were designed and synthesized as internal standards. Validation of the method showed that it has good sensitivity, specificity, reproducibility, precision, and accuracy. This method is easily applicable in routine laboratory analysis without intensive proteomics background. [ABSTRACT FROM AUTHOR]

In contrast to quantification of biotherapeutics, endogenous protein biomarker and target quantification using LC-MS based targeted proteomics can require a much more stringent and time-consuming tryptic signature peptide selection for each specific application. While some general criteria exist, there are no tools currently available in the public domain to predict the ionization efficiency for a given signature peptide candidate. Lack of knowledge of the ionization efficiencies forces investigators to choose peptides blindly, thus hindering method development for low abundant protein quantification. Here, the authors propose a tryptic signature peptide selection workflow to achieve a more efficient method development and to improve success rates in signature peptide selection for low abundant endogenous target and protein biomarker quantification.

Background: Pinelliae Rhizoma (PR), a toxic medication, with long history, is commonly used for eliminating phlegm. Due to the shortage of wild resources and the relative lacking of cultivation technology, it is often confused with its counterfeit species in the market, such as Typhonii Rhizoma (TR), Arisaematis Rhizoma (AR) and tubers of Typhonium flagelliforme (TF) and Pinellia pedatisecta (PP).Purpose: It was aimed to screen signature enzymatic peptides from toxic proteins to identify PR and its four counterfeit species.Study Design: A comparative proteogenomics strategy based on open-source transcriptome data was applied for screening signature peptides from toxic proteins, which were applied for species authentication of PR and its counterfeit species.Methods: Firstly, the open-source transcriptome data was used for constructing the annotated protein database, which was used for peptides identification. Secondly, the toxicity of different fractions of PR were evaluated by the rat peritoneal inflammation model. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to profile the main proteins bands of five species, whose sequences were identified based on the in-gel digestion experiment by using ultra-high-performance liquid chromatography/quadrupole-Orbitrap mass spectrometry. Finally, the label-free proteomic analysis was performed to character the proteins and screen the signature peptides of five species, which were validated in commercially available products by dynamic multi reaction monitor (DMRM).Results: The results in this study confirmed that protein was the main toxic components of PR. Both Pinellia ternata agglutinin (PTA) and trypsin inhibitor (TI) like proteins are the main proteins, which were characterized by proteomic analysis based on four annotated protein database. Meanwhile, seven signature peptides from toxic proteins were screened and validated with good repeatability and specificity in commercial products.Conclusion: Seven signature enzymatic peptides from toxic protein screened by the comparative proteogenomics strategy based on open-source transcriptome data achieved good identification ability of PR and its four counterfeit species. [ABSTRACT FROM AUTHOR]

Abstract: Quantitative analysis of a therapeutic protein through use of surrogate proteotypic peptides was evaluated for the measurement of Amevive (Alefacept) in human plasma using liquid chromatography tandem mass spectrometry. Signature peptides were obtained through in silico and iterative tuning processes to represent Alefacept for quantification. Horse heart myoglobin was chosen as a protein analogue internal standard to compensate for errors associated with matrix effects and to track recovery throughout the entire sample pretreatment process. Samples were prepared for analysis by selective precipitation of the target proteins with pH controlled at 5.1 and heat denaturation at 45°C followed by enzymatic digestion, dilution, and filtration. On-line extraction of the signature peptides was carried out using a Phenomenex Gemini C18 security guard column (4.0mm×2.0mm) as a loading column and a Gemini C18 (100mm×2.1 I.D., particle size 5μm) as the analytical (eluting) column. Tandem mass spectrometric detection was performed on a hybrid triple quadrupole linear ion trap equipped with electrospray ionization to positively ionize signature peptides for Alefacept and myoglobin. The method was linear for Alefacept (protein) concentrations between 250 and 10,000ng/mL. Precision and accuracy for inter- and intra-assay for the lower limit of quantification was less than 20% (16.2 and 10.3, respectively). The method was validated according to current FDA guidelines for bioanalytical method validation. [Copyright &y& Elsevier]

An innovative analytical strategy has been applied to identify signature peptides able to distinguish among processed animal proteins (PAPs) derived from bovine, pig, fish, and milk products. Proteomics was first used to elucidate the proteome of each source. Starting from the identified proteins and using a funnel based approach, a set of abundant and well characterized peptides with suitable physical-chemical properties (signature peptides) and specific for each source was selected. An on-target LC-ESI-MS/MS method (MRM mode) was set up using standard peptides and was then applied to selectively identify the PAP source and also to distinguish proteins from bovine carcass and milk proteins. We believe that the method described meets the request of the European Commission which has developed a strategy for gradually lifting the "total ban" toward "species to species ban", therefore requiring official methods for species-specific discrimination in feed.

Aim: Immobilized metal ion affinity chromatography is widely employed for purifying polyhistidine-tagged recombinant proteins from cell lysates. The technique can be applied for quantification of therapeutic proteins in biological matrices by LC-MS/MS., Results: A protein reagent-free workflow was developed for quantifying polyhistidine-tagged proteins by LC-MS/MS. The workflow includes target protein enrichment by immobilized metal ion affinity chromatography, on-bead trypsin digestion and quantification of signature peptides by LC-MS/MS. It was applied to quantify a 6×His-tagged protein in a mouse pharmacokinetic study with assay sensitivity of 10.0 ng/ml and linearity up to 10,000 ng/ml., Conclusion: The protein reagent-free workflow developed herein can overcome reagent limitation and serve as a viable approach for quantifying polyhistidine-tagged therapeutic proteins to support discovery pharmacokinetic and pharmacodynamic studies.

The term "proteome" was first introduced into the scientific literature in July 1995. Almost 20 years ago attempts to characterize the "total protein complement able to be encoded by a given genome" only became possible due to privileged access to what were then the world's most complete sets of genomic data. Today, proteomics has become an important pillar in the fields of disease diagnosis and drug research and development, while also playing a critical role in the much larger field of Healthcare Analytics and Biomarker Discovery and Detection. It is important to note that this industry originated mostly from building blocks in analytical science that predated the term "proteomics" by many decades. However, proteomics, as a discipline, has allowed protein scientists to more favorably compete in the face of highly fashionable Big Science and, more specifically, genomics., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)