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1.
Mol Cell Proteomics ; 11(6): M111.013854, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22199228

RESUMO

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 µl 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-MRM-MS can be made reproducible across independent laboratories and has the potential to be adopted widely for assaying proteins in matrices as complex as plasma.


Assuntos
Proteínas Sanguíneas/química , Proteínas Sanguíneas/isolamento & purificação , Animais , Automação Laboratorial , Cromatografia de Afinidade/normas , Fator Estimulador de Colônias de Granulócitos/sangue , Humanos , Imunoprecipitação/normas , Limite de Detecção , Fragmentos de Peptídeos/química , Coelhos , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/normas
2.
Mol Cell Proteomics ; 10(4): M110.005645, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21245105

RESUMO

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/µl 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.


Assuntos
Proteínas Sanguíneas/imunologia , Proteoma/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Sanguíneas/metabolismo , Proteínas de Transporte/sangue , Proteínas de Transporte/imunologia , Humanos , Soros Imunes , Imunoensaio/métodos , Espectrometria de Massas/métodos , Proteínas dos Microfilamentos/sangue , Proteínas dos Microfilamentos/imunologia , Técnicas de Diagnóstico Molecular , Peptídeos/imunologia , Coelhos , Sensibilidade e Especificidade
3.
J Proteome Res ; 11(12): 5642-9, 2012 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-23126378

RESUMO

We investigated the utility of an SPE-MS/MS platform in combination with a modified SISCAPA workflow for chromatography-free MRM analysis of proteotypic peptides in digested human plasma. This combination of SISCAPA and SPE-MS/MS technology allows sensitive, MRM-based quantification of peptides from plasma digests with a sample cycle time of ∼7 s, a 300-fold improvement over typical MRM analyses with analysis times of 30-40 min that use liquid chromatography upstream of MS. The optimized system includes capture and enrichment to near purity of target proteotypic peptides using rigorously selected, high affinity, antipeptide monoclonal antibodies and reduction of background peptides using a novel treatment of magnetic bead immunoadsorbents. Using this method, we have successfully quantitated LPS-binding protein and mesothelin (concentrations of ∼5000 ng/mL and ∼10 ng/mL, respectively) in human plasma. The method eliminates the need for upstream liquid-chromatography and can be multiplexed, thus facilitating quantitative analysis of proteins, including biomarkers, in large sample sets. The method is ideal for high-throughput biomarker validation after affinity enrichment and has the potential for applications in clinical laboratories.


Assuntos
Proteínas Sanguíneas/análise , Ensaios de Triagem em Larga Escala/métodos , Espectrometria de Massas/métodos , Peptídeos/sangue , Software , Proteínas de Fase Aguda/análise , Anticorpos Monoclonais/química , Afinidade de Anticorpos , Biomarcadores/sangue , Proteínas de Transporte/análise , Cromatografia Líquida , Proteínas Ligadas por GPI/sangue , Humanos , Glicoproteínas de Membrana/análise , Mesotelina , Proteômica/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de Tempo
4.
Elife ; 102021 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-34747696

RESUMO

Reliable, robust, large-scale molecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for monitoring the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a scalable analytical approach to detect viral proteins based on peptide immuno-affinity enrichment combined with liquid chromatography-mass spectrometry (LC-MS). This is a multiplexed strategy, based on targeted proteomics analysis and read-out by LC-MS, capable of precisely quantifying and confirming the presence of SARS-CoV-2 in phosphate-buffered saline (PBS) swab media from combined throat/nasopharynx/saliva samples. The results reveal that the levels of SARS-CoV-2 measured by LC-MS correlate well with their correspondingreal-time polymerase chain reaction (RT-PCR) read-out (r = 0.79). The analytical workflow shows similar turnaround times as regular RT-PCR instrumentation with a quantitative read-out of viral proteins corresponding to cycle thresholds (Ct) equivalents ranging from 21 to 34. Using RT-PCR as a reference, we demonstrate that the LC-MS-based method has 100% negative percent agreement (estimated specificity) and 95% positive percent agreement (estimated sensitivity) when analyzing clinical samples collected from asymptomatic individuals with a Ct within the limit of detection of the mass spectrometer (Ct ≤ 30). These results suggest that a scalable analytical method based on LC-MS has a place in future pandemic preparedness centers to complement current virus detection technologies.


Assuntos
COVID-19/diagnóstico , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Técnicas de Diagnóstico Molecular/métodos , Proteínas Virais/análise , COVID-19/virologia , Humanos , Modelos Lineares , Nasofaringe/virologia , Fragmentos de Peptídeos/análise , Proteômica , Reprodutibilidade dos Testes , SARS-CoV-2/química , Sensibilidade e Especificidade
5.
Bioanalysis ; 12(13): 937-955, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32253915

RESUMO

Aim: High-frequency longitudinal tracking of inflammation using dried blood microsamples provides a new window for personalized monitoring of infections, chronic inflammatory disease and clinical trials of anti-inflammatory drugs. Results/methodology: Using 1662 dried blood spot samples collected by 16 subjects over periods of weeks to years, we studied the behavior of 12 acute phase response and related proteins in inflammation events correlated with infection, vaccination, surgery, intense exercise and Crohn's disease. Proteins were measured using SISCAPA mass spectrometry and normalized to constant plasma volume using low-variance proteins, generating high precision within-person biomarker trajectories with well-characterized personal baselines. Discussion/conclusion: The results shed new light on the dynamic regulation of APR responses, offering a new approach to visualization of multidimensional inflammation trajectories.


Assuntos
Teste em Amostras de Sangue Seco/métodos , Adulto , Idoso , Feminino , Humanos , Inflamação/sangue , Masculino , Pessoa de Meia-Idade , Fatores de Tempo
6.
J Immunol Methods ; 341(1-2): 86-96, 2009 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-19041872

RESUMO

A refined surface plasmon resonance method was developed to measure the kinetics of peptide binding to rabbit monoclonal antibodies (RabMAbs). Optimized amounts of RabMAbs were captured onto sensor chips from hybridoma supernatants followed by binding of free peptides from solution. This allowed kinetic measurement of monovalent interactions of peptides with single antigen binding sites on the antibodies and determination of affinity constants without complications contributed by avidity considerations. Peptide-binding responses were normalized for the amount of antibody present in each sample and a simple interaction model was fit to all of the binding responses simultaneously. As a result, the kinetic rate constants ka and kd, and the affinity constant KD (kd/ka), could be determined for each antibody interaction under identical conditions. Higher-resolution studies involving multiple concentrations of peptide antigens were performed to validate the reliability of single-concentration measurements. By combining data on affinity, activity and concentration, ranking of the antibody-containing supernatants was performed, allowing selection of high quality RabMAbs for binding of peptides in solution.


Assuntos
Anticorpos Monoclonais/química , Afinidade de Anticorpos/fisiologia , Peptídeos/química , Ressonância de Plasmônio de Superfície/métodos , Animais , Anticorpos Monoclonais/imunologia , Humanos , Hibridomas/citologia , Hibridomas/imunologia , Cinética , Peptídeos/imunologia , Coelhos
7.
Bioanalysis ; 8(15): 1597-1609, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27420772

RESUMO

BACKGROUND: The use of DBS for quantitative protein biomarker measurement has been hindered by issues associated with blood hematocrit variations and lack of detection sensitivity, particularly when multiple biomarkers are measured. MATERIALS & METHODS: An automated, multiplexed SISCAPA analysis was used to normalize blood volume variations in DBS and quantify proteins of varying abundance in longitudinal specimens. CONCLUSION: The results showed that after normalizing the spot-to-spot hematocrit variations, peptide surrogates of protein biomarkers could be accurately quantitated in DBS. This allowed the establishment of baselines for a variety of biomarkers in multiple individuals and enabled detection of changes over time, thus offering an effective solution for longitudinal personal monitoring of biomarkers relevant in health and disease.


Assuntos
Proteínas Sanguíneas/análise , Teste em Amostras de Sangue Seco/métodos , Biomarcadores/análise , Biomarcadores/sangue , Volume Sanguíneo , Cromatografia Líquida/métodos , Hematócrito , Humanos , Espectrometria de Massas em Tandem/métodos , Fluxo de Trabalho
8.
N Biotechnol ; 33(5 Pt A): 494-502, 2016 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-26772726

RESUMO

Efficient robotic workflows for trypsin digestion of human plasma and subsequent antibody-mediated peptide enrichment (the SISCAPA method) were developed with the goal of improving assay precision and throughput for multiplexed protein biomarker quantification. First, an 'addition only' tryptic digestion protocol was simplified from classical methods, eliminating the need for sample cleanup, while improving reproducibility, scalability and cost. Second, methods were developed to allow multiplexed enrichment and quantification of peptide surrogates of protein biomarkers representing a very broad range of concentrations and widely different molecular masses in human plasma. The total workflow coefficients of variation (including the 3 sequential steps of digestion, SISCAPA peptide enrichment and mass spectrometric analysis) for 5 proteotypic peptides measured in 6 replicates of each of 6 different samples repeated over 6 days averaged 3.4% within-run and 4.3% across all runs. An experiment to identify sources of variation in the workflow demonstrated that MRM measurement and tryptic digestion steps each had average CVs of ∼2.7%. Because of the high purity of the peptide analytes enriched by antibody capture, the liquid chromatography step is minimized and in some cases eliminated altogether, enabling throughput levels consistent with requirements of large biomarker and clinical studies.


Assuntos
Proteínas Sanguíneas/análise , Automação , Biomarcadores/análise , Biotecnologia , Proteínas Sanguíneas/química , Proteínas Sanguíneas/imunologia , Humanos , Peso Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Proteômica/métodos , Reprodutibilidade dos Testes , Robótica , Espectrometria de Massas em Tandem/métodos , Tripsina , Fluxo de Trabalho
9.
PLoS Negl Trop Dis ; 10(4): e0004510, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27055052

RESUMO

BACKGROUND: Identification of species-specific trypanosome molecules is important for laboratory- and field-based research into epidemiology and disease diagnosis. Although Trypanosoma congolense is the most important trypanosome pathogen of cattle in Africa, no species-specific molecules found in infective bloodstream forms (BSF) of the parasites have been identified, thus limiting development of diagnostic tests. METHODS: Immuno-mass spectrometric methods were used to identify a protein that is recognized by a T. congolense-specific monoclonal antibody (mAb) Tc6/42.6.4. The identified molecule was expressed as a recombinant protein in E. coli and was tested in several immunoassays for its ability to interact with the mAb. The three dimensional structure of the protein was modeled and compared to crystal- and NMR-structures of the homologous proteins from T. cruzi and T. brucei respectively, in order to examine structural differences leading to the different immunoreactivity of the T. congolense molecule. Enzyme-linked immunosorbent assays (ELISA) were used to measure antibodies produced by trypanosome-infected African cattle in order to assess the potential for use of T. congolense calflagin in a serodiagnostic assay. RESULTS: The antigen recognized by the T. congolense-specific mAb Tc6/42.6.4 was identified as a flagellar calcium-binding protein, calflagin. The recombinant molecule showed immunoreactivity with the T. congolense-specific mAb confirming that it is the cognate antigen. Immunofluorescence experiments revealed that Ca2+ modulated the localization of the calflagin molecule in trypanosomes. Structural modelling and comparison with calflagin homologues from other trypanosomatids revealed four non-conserved regions on the surface of the T. congolense molecule that due to differences in surface chemistry and structural topography may form species-specific epitopes. ELISAs using the recombinant calflagin as antigen to detect antibodies in trypanosome-infected cattle showed that the majority of cattle had antibody responses. Area under the Receiver-Operating Characteristic (ROC) curves, associated with host IgG and IgM, were calculated to be 0.623 and 0.709 respectively, indicating a positive correlation between trypanosome infection and the presence of anti-calflagin antibodies. CONCLUSIONS: While calflagin is conserved among different species of African trypanosomes, our results show that T. congolense calflagin possesses unique epitopes that differentiate this protein from homologues in other trypanosome species. MAb Tc6/42.6.4 has clear utility as a laboratory tool for identifying T. congolense. T. congolense calflagin has potential as a serodiagnostic antigen and should be explored further for its utility in antigen-detection assays for diagnosis of cattle infections.


Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Trypanosoma congolense/química , Animais , Anticorpos Monoclonais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Bovinos , Ensaio de Imunoadsorção Enzimática/métodos , Mapeamento de Epitopos , Escherichia coli/genética , Espectrometria de Massas , Modelos Moleculares , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Testes Sorológicos , Trypanosoma brucei brucei/química , Trypanosoma congolense/imunologia , Tripanossomíase Bovina/diagnóstico , Tripanossomíase Bovina/imunologia
10.
J Immunol Methods ; 364(1-2): 50-64, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21078325

RESUMO

A scalable method for screening and selection of peptide-specific monoclonal antibodies (mAbs) is described. To identify high affinity anti-peptide mAbs in hybridoma supernatants, antibodies were captured by magnetic affinity beads followed by binding of specific peptides from solution. After timed washing steps, the remaining bound peptides were eluted from the beads and detected by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). This allowed measurement of monovalent interactions of peptides with single antigen binding sites on the antibodies, thus reflecting antibody affinity rather than avidity. Antibodies that were able to bind target peptides from solution phase and retain them during washing for a minimum of 10 min were identified by the strength of the appropriate m/z peptide MS signals obtained. This wash time reflects the minimum peptide dissociation time required for use of these antibodies in several current immuno-mass spectrometry assays. Kinetic analysis of antibody-peptide binding by surface plasmon resonance (SPR) showed that the selected antibodies were of high affinity and, most importantly, had low dissociation constants. This method, called MALDI immunoscreening (MiSCREEN), thus enables rapid screening and selection of high affinity anti-peptide antibodies that are useful for a variety of immunoproteomics applications. To demonstrate their functional utility in immuno-mass spectrometry assays, we used the selected, purified RabMAbs to enrich natural (tryptic) peptides from digested human plasma.


Assuntos
Anticorpos Monoclonais/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Ensaios de Triagem em Larga Escala , Humanos , Hibridomas/metabolismo , Técnicas de Imunoadsorção , Camundongos , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/imunologia , Coelhos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ressonância de Plasmônio de Superfície
11.
J Vis Exp ; (53)2011 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-21841765

RESUMO

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').(1) An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).(2) In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules (3, 4) and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.(5-7) 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 enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.(8-13) In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.


Assuntos
Imunoensaio/métodos , Espectrometria de Massas/métodos , Peptídeos/análise , Animais , Camundongos , Osteopontina/análise , Fragmentos de Peptídeos/análise
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