RESUMO
Recombinant immunoglobulins (rIgGs) have become increasingly important as therapeutic agents and diagnostic tools in recent years. Genetic engineering allows the introduction of non-natural features such as the Sortase motif for site-directed labeling. In this study, the enzyme Sortase A (SrtA) was used for the proteolytic cleavage of rIgGs to produce their biotinylated Fab fragments by locating the cleavage site close to the hinge region. However, SrtA cleavage of engineered rabbit IgGs (rRb-IgGs) derived from human embryonic kidney (HEK) 293 cells showed significantly lower yields compared with their mouse counterparts. Nonrecombinant Rb-IgGs have N- and O-glycans, and the presence of O-glycans close to the hinge region of the rRb-IgGs might affect the susceptibility of these antibodies to SrtA cleavage. In addition, the glycosylation pattern of rIgGs differs depending on the host cell used for expression. Therefore, we analyzed the N- and O-glycans of various rRb-IgGs expressed in HEK293 cells, detecting and quantifying 13 different N-glycan and 3 different O-glycan structures. The distribution of the different detected glycoforms in our rRb-IgG N-glycan analysis is in agreement with previous studies on recombinant human IgG N-glycans, confirming the hypothesis that the host cell defines the glycosylation of the recombinant produced IgGs. O-glycosylation could be mapped onto the threonine residue within the hinge region sequence XPTCPPPX, as already described previously for nonrecombinant Rb-IgGs. Substitution of this threonine allowed an almost complete Fab fragment cleavage. Therefore, we could confirm the hypothesis that the O-glycans affect the SrtA activity, probably due to steric hindrance.
Assuntos
Imunoglobulina G , Peptídeo Hidrolases , Animais , Glicosilação , Células HEK293 , Humanos , Imunoglobulina G/química , Camundongos , Polissacarídeos/química , CoelhosRESUMO
INTRODUCTION: Reference materials based on human cerebrospinal fluid were certified for the mass concentration of amyloid beta (Aß)1-42 (Aß42 ). They are intended to be used to calibrate diagnostic assays for Aß42 . METHODS: The three certified reference materials (CRMs), ERM-DA480/IFCC, ERM-DA481/IFCC and ERM-DA482/IFCC, were prepared at three concentration levels and characterized using isotope dilution mass spectrometry methods. Roche, EUROIMMUN, and Fujirebio used the three CRMs to re-calibrate their immunoassays. RESULTS: The certified Aß42 mass concentrations in ERM-DA480/IFCC, ERM-DA481/IFCC, and ERM-DA482/IFCC are 0.45, 0.72, and 1.22 µg/L, respectively, with expanded uncertainties (k = 2) of 0.07, 0.11, and 0.18 µg/L, respectively. Before re-calibration, a good correlation (Pearson's r > 0.97), yet large biases, were observed between results from different commercial assays. After re-calibration the between-assay bias was reduced to < 5%. DISCUSSION: The Aß42 CRMs can ensure the equivalence of results between methods and across platforms for the measurement of Aß42 .
Assuntos
Peptídeos beta-Amiloides/líquido cefalorraquidiano , Imunoensaio/normas , Calibragem , Humanos , Imunoensaio/métodos , Padrões de ReferênciaRESUMO
Prostate cancer represents a major cause of cancer death in men worldwide. Novel non-invasive methods are still required for differentiation of non-aggressive from aggressive tumors. Recently, changes in prostate-specific antigen glycosylation pattern, such as core-fucosylation, have been described in prostate cancer. The objective of this study was to evaluate whether the core-fucosylation determinant of serum prostate-specific antigen may serve as refined marker for differentiation between benign prostate hyperplasia and prostate cancer or identification of aggressive prostate cancer. A previously developed liquid chromatography-mass spectrometry/mass spectrometry-based strategy was used for multiplex analysis of core-fucosylated prostate-specific antigen (fuc-PSA) and total prostate-specific antigen levels in sera from 50 benign prostate hyperplasia and 100 prostate cancer patients of different aggressiveness (Gleason scores, 5-10) covering the critical gray area (2-10 ng/mL). For identification of aggressive prostate cancer, the ratio of fuc-PSA to total prostate-specific antigen (%-fuc-PSA) yielded a 5%-8% increase in the area under the curve (0.60) compared to the currently used total prostate-specific antigen (area under the curve = 0.52) and %-free prostate-specific antigen (area under the curve = 0.55) tests. However, our data showed that aggressive prostate cancer (Gleason score > 6) and non-aggressive prostate cancer (Gleason score ≤ 6) could not significantly (p-value = 0.08) be differentiated by usage of %-fuc-PSA. In addition, both non-standardized fuc-PSA and standardized %-fuc-PSA had no diagnostic value for differentiation of benign prostate hyperplasia from prostate cancer. The %-fuc-PSA serum levels could not improve the differentiation of non-aggressive and aggressive prostate cancer compared to conventional diagnostic prostate cancer markers. Still, it is unclear whether these limitations come from the biomarker, the used patient cohort, or the imprecision of the applied method itself. Therefore, %-fuc-PSA should be further investigated, especially by more precise methods whether it could be clinically used in prostate cancer diagnosis.
Assuntos
Biomarcadores Tumorais/química , Antígeno Prostático Específico/química , Hiperplasia Prostática/diagnóstico , Hiperplasia Prostática/patologia , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/patologia , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Cromatografia Líquida , Diagnóstico Diferencial , Glicosilação , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Próstata/patologia , Antígeno Prostático Específico/sangue , Espectrometria de Massas em TandemRESUMO
BACKGROUND: The core Alzheimer's disease cerebrospinal fluid (CSF) biomarkers total tau (T-tau), phosphorylated tau (P-tau), ß-amyloid 1-42 (Aß42) and ß-amyloid 1-40 (Aß40) are increasing in importance and are now part of the research criteria for the diagnosis of the disease. The main aim of this study is to evaluate whether a set of certified reference materials (CRMs) are commutable for Aß42 and to serve as a feasibility study for the other markers. This property is a prerequisite for the establishment of CRMs which will then be used by manufacturers to calibrate their assays against. Once the preanalytical factors have been standardized and proper selection criteria are available for subject cohorts this harmonization between methods will allow for universal cut-offs to be determined. METHODS: Thirty-four individual CSF samples and three different CRMs where analyzed for T-tau, P-tau, Aß42 and Aß40, using up to seven different commercially available methods. For Aß40 and Aß42 a mass spectrometry-based procedure was also employed. RESULTS: There were strong pairwise correlations between the different methods (Spearman's ρ>0.92) for all investigated analytes and the CRMs were not distinguishable from the individual samples. CONCLUSIONS: This study shows that the CRMs are commutable for the different assays for Aß42. For the other analytes the results show that it would be feasible to also produce CRMs for these. However, additional studies are needed as the concentration interval for the CRMs were selected based on Aß42 concentrations only and did in general not cover satisfactory large concentration intervals for the other analytes.
Assuntos
Peptídeos beta-Amiloides/líquido cefalorraquidiano , Imunoensaio/normas , Fragmentos de Peptídeos/líquido cefalorraquidiano , Proteínas tau/líquido cefalorraquidiano , Biomarcadores/líquido cefalorraquidiano , Humanos , Padrões de ReferênciaRESUMO
INTRODUCTION: Available assays for quantitation of the Alzheimer's disease (AD) biomarker amyloid-beta 1-42 (Aß [1-42]) in cerebrospinal fluid demonstrate significant variability and lack of standardization to reference measurement procedures (RMPs). We report analytical performance data for the novel Elecsys ß-amyloid (1-42) assay (Roche Diagnostics). METHODS: Lot-to-lot comparability was tested using method comparison. Performance parameters were measured according to Clinical & Laboratory Standards Institute (CLSI) guidelines. The assay was standardized to a Joint Committee for Traceability in Laboratory Medicine (JCTLM) approved RMP. RESULTS: Limit of quantitation was <11.28 pg/mL, and the assay was linear throughout the measuring range (200-1700 pg/mL). Excellent lot-to-lot comparability was observed (correlation coefficients [Pearson's r] >0.995; bias in medical decision area <2%). Repeatability coefficients of variation (CVs) were 1.0%-1.6%, intermediate CVs were 1.9%-4.0%, and intermodule CVs were 1.1%-3.9%. Estimated total reproducibility was 2.0%-5.1%. Correlation with the RMP was good (Pearson's r, 0.93). DISCUSSION: The Elecsys ß-amyloid (1-42) assay has high analytical performance that may improve biomarker-based AD diagnosis.
Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Peptídeos beta-Amiloides , Biomarcadores/líquido cefalorraquidiano , Imunoensaio/normas , Luminescência , Fragmentos de Peptídeos , Biomarcadores/análise , Humanos , Imunoensaio/instrumentação , Imunoensaio/métodos , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Cerebrospinal fluid (CSF) amyloid-ß (Aß42) is a well-established biomarker for Alzheimer disease. Several immunoassays for Aß42 exist but differ in absolute concentrations and may suffer from matrix interference, thereby hampering interlaboratory comparisons and the use of general cutoff levels. Together with the IFCC Working Group on CSF Proteins, we developed a candidate reference measurement procedure (RMP) for Aß42. METHODS: The antibody-independent candidate RMP was based on solid-phase extraction and isotope-dilution LC-MS/MS. The candidate RMP used 2 differently stable isotope-labeled Aß42 peptides for calibration in human CSF, an important aspect since there was no analyte-free matrix available. Because no CSF certified reference material (CRM) exists, we used a nonlabeled Aß42 standard, the concentration of which was determined by amino acid analysis. We performed measurements on a high-resolution quadrupole-Orbitrap hybrid instrument. The results were compared with a method run in a second laboratory with triple quadrupole instrumentation. RESULTS: The candidate RMP allowed quantification of CSF Aß42 from 150 to 4000 pg/mL. Validation of the method showed a recovery of 100% (15%), intraassay and interassay imprecision of 5.0% and 6.4%, respectively, and an expanded uncertainty of 15.7%. No analytical interferences or carryover were detected. CONCLUSIONS: This method will help set the value of CSF Aß42 in a CRM, which could be used to harmonize Aß42 assays and facilitate the introduction of general cutoff concentrations for CSF Aß42 in clinical trials and practice.
Assuntos
Peptídeos beta-Amiloides/líquido cefalorraquidiano , Fragmentos de Peptídeos/líquido cefalorraquidiano , Biomarcadores/líquido cefalorraquidiano , Calibragem , Isótopos de Carbono , Cromatografia Líquida/normas , Humanos , Isótopos de Nitrogênio , Padrões de Referência , Espectrometria de Massas em Tandem/normasRESUMO
BACKGROUND: The aim of our work was to develop and validate a reliable LC-MS/MS-based measurement procedure for the quantification of vancomycin in serum, to be applied in the context of efforts to standardize and harmonize therapeutic drug monitoring of this compound using routine assays. METHODS: Sample preparation was based on protein precipitation followed by ultrafiltration. In order to minimize differential modulation of ionization by matrix constituents extended chromatographic separation was applied leading to a retention time of 9.8 min for the analyte. Measurement was done by HPLC-ESI-MS/MS. For internal standardization the derivative vancomycin-glycin (ISTD) prepared by chemical synthesis was used, HPLC conditions ensured coelution of ISTD with the analyte. RESULTS: In a bi-center validation total CVs of <4% were observed for quality control material ranging from 5.3 mg/L to 79.4 mg/L; accuracy was ±4%. No relevant ion suppression was observed. Comparative measurement of aliquots from 70 samples at the two validation sites demonstrated close agreement. CONCLUSIONS: Employing a closely related homologue molecule for internal standardization and the use of MS/MS following highly efficient sample pre-fractionation by HPLC, the method described here can be considered to offer the highest level of analytical reliability realized so far for the quantification of vancomycin in human serum. Thus, the method is suitable to be used in a comprehensive reference measurement system for vancomycin.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas em Tandem/métodos , Vancomicina/sangue , Antibacterianos/sangue , Cromatografia Líquida de Alta Pressão/normas , Humanos , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/normasRESUMO
De novo sequencing of peptides using tandem MS is difficult due to missing fragment ions in the spectra commonly obtained after CID of peptide precursor ions. Complementing CID spectra with spectra obtained in an ion-trap mass spectrometer upon electron transfer dissociation (ETD) significantly increases the sequence coverage with diagnostic ions. In the de novo sequencing algorithm CompNovo presented here, a divide-and-conquer approach was combined with an efficient mass decomposition algorithm to exploit the complementary information contained in CID and ETD spectra. After optimizing the parameters for the algorithm on a well-defined training data set obtained for peptides from nine known proteins, the CompNovo algorithm was applied to the de novo sequencing of peptides derived from a whole protein extract of Sorangium cellulosum bacteria. To 2406 pairs of CID and ETD spectra contained in this data set, 675 fully correct sequences were assigned, which represent a success rate of 28.1%. It is shown that the CompNovo algorithm yields significantly improved sequencing accuracy as compared with published approaches using only CID spectra or combined CID and ETD spectra.
Assuntos
Proteínas de Bactérias/química , Myxococcales/química , Peptídeos/química , Análise de Sequência de Proteína/métodos , Espectrometria de Massas em Tandem/métodos , Algoritmos , Proteoma/análiseRESUMO
Anti-tumor immunity is limited by a number of factors including the lack of fully activated T-cells, insufficient antigenic stimulation and the immune-suppressive tumor microenvironment. We addressed these hurdles by developing a novel class of immunoconjugates, Antibody-Targeted Pathogen-derived Peptides (ATPPs), which were designed to efficiently deliver viral T-cell epitopes to tumors with the aim of redirecting virus-specific memory T-cells against the tumor. ATPPs were generated through covalent binding of mature MHC class I peptides to antibodies specific for cell surface-expressed tumor antigens that mediate immunoconjugate internalization. By means of a cleavable linker, the peptides are released in the endosomal compartment, from which they are loaded into MHC class I without the need for further processing. Pulsing of tumor cells with ATPPs was found to sensitize these for recognition by virus-specific CD8+ T-cells with much greater efficiency than exogenous loading with free peptides. Systemic injection of ATPPs into tumor-bearing mice enhanced the recruitment of virus-specific T-cells into the tumor and, when combined with immune checkpoint blockade, suppressed tumor growth. Our data thereby demonstrate the potential of ATPPs as a means of kick-starting the immune response against "cold" tumors and increasing the efficacy of checkpoint inhibitors.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/administração & dosagem , Imunoconjugados/administração & dosagem , Neoplasias/terapia , Peptídeos/administração & dosagem , Animais , Linhagem Celular Tumoral , Feminino , Herpesvirus Humano 4 , Humanos , Imunoterapia , CamundongosRESUMO
BACKGROUND: Alzheimer's disease (AD) biomarkers, such as cerebrospinal fluid (CSF) amyloid-ß (1-42; Aß42), can provide high diagnostic accuracy. Several immunoassays are available for Aß42 quantitation, but standardisation across assays remains an issue. We compared the Elecsys® ß-Amyloid (1-42) CSF assay with three assays and two liquid chromatography tandem mass spectrometry (LC-MS/MS) methods. METHODS: Three method comparison studies evaluated the correlation between the Elecsys® ß-Amyloid (1-42) CSF assay versus: INNOTEST® ß-AMYLOID(1-42) (860 samples) and the Roche Diagnostics-developed LC-MS/MS method (250 samples); INNO-BIA AlzBio3 and the University of Pennsylvania (UPenn)-developed LC-MS/MS method (250 samples); and ADx-EUROIMMUN Beta-Amyloid (1-42) enzyme-linked immunosorbent assay (ELISA) (49 samples). RESULTS: High correlation was demonstrated between Elecsys® ß-Amyloid (1-42) CSF and comparator assays: INNOTEST® ß-AMYLOID(1-42) (Spearman's ρ, 0.954); INNO-BIA AlzBio3 (Spearman's ρ, 0.864); ADx-EUROIMMUN Beta-Amyloid (1-42) ELISA (Pearson's r, 0.925). Elecsys® assay and LC-MS/MS measurements were highly correlated: Pearson's r, 0.949 (Roche Diagnostics-developed method) and 0.943 (UPenn-developed method). CONCLUSION: Findings from this multicentre evaluation further support use of the Elecsys® ß-Amyloid (1-42) CSF assay to aid AD diagnosis. CSF-based certified reference materials should improve agreement across assays and mass spectrometry-based methods, which is essential to establish a global uniform CSF Aß42 cut-off to detect amyloid pathology.
Assuntos
Peptídeos beta-Amiloides/líquido cefalorraquidiano , Técnicas Eletroquímicas/métodos , Imunoensaio/métodos , Fragmentos de Peptídeos/líquido cefalorraquidiano , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/diagnóstico , Biomarcadores/líquido cefalorraquidiano , Cromatografia Líquida/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Espectrometria de Massas em Tandem/métodosRESUMO
MOTIVATION: Liquid chromatography coupled to mass spectrometry (LC-MS) and combined with tandem mass spectrometry (LC-MS/MS) have become a prominent tool for the analysis of complex proteomic samples. An important step in a typical workflow is the combination of results from multiple LC-MS experiments to improve confidence in the obtained measurements or to compare results from different samples. To do so, a suitable mapping or alignment between the data sets needs to be estimated. The alignment has to correct for variations in mass and elution time which are present in all mass spectrometry experiments. RESULTS: We propose a novel algorithm to align LC-MS samples and to match corresponding ion species across samples. Our algorithm matches landmark signals between two data sets using a geometric technique based on pose clustering. Variations in mass and retention time are corrected by an affine dewarping function estimated from matched landmarks. We use the pairwise dewarping in an algorithm for aligning multiple samples. We show that our pose clustering approach is fast and reliable as compared to previous approaches. It is robust in the presence of noise and able to accurately align samples with only few common ion species. In addition, we can easily handle different kinds of LC-MS data and adopt our algorithm to new mass spectrometry technologies. AVAILABILITY: This algorithm is implemented as part of the OpenMS software library for shotgun proteomics and available under the Lesser GNU Public License (LGPL) at www.openms.de.
Assuntos
Algoritmos , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Mapeamento de Peptídeos/métodos , Proteoma/química , Alinhamento de Sequência/métodos , Análise de Sequência de Proteína/métodos , Sequência de AminoácidosRESUMO
Recently, site-specific fucosylation of glycoproteins has attracted attention as it can be associated with several types of cancers including prostate cancer. However, individual glycoproteins, which might serve as potential cancer markers, often are very low-concentrated in complex serum matrices and distinct glycan structures are hard to detect by immunoassays. Here, we present a mass spectrometry-based strategy for the simultaneous analysis of core-fucosylated and total prostate-specific antigen (PSA) in human serum in the low ng/ml concentration range. Sample preparation comprised an immunoaffinity capture step to enrich total PSA from human serum using anti-PSA antibody coated magnetic beads followed by consecutive two-step on-bead partial deglycosylation with endoglycosidase F3 and tryptic digestion prior to LC-MS/MS analysis. The method was shown to be linear from 0.5 to 60â¯ng/ml total PSA concentrations and allows the simultaneous quantification of core-fucosylated PSA down to 1â¯ng/ml and total PSA lower than 0.5â¯ng/ml. The imprecision of the method over two days ranged from 9.7-23.2% for core-fucosylated PSA and 10.3-18.3% for total PSA depending on the PSA level. The feasibility of the method in native sera was shown using three human specimens. To our knowledge, this is the first MS-based method for quantification of core-fucosylated PSA in the low ng/ml concentration range in human serum. This method could be used in large patient cohorts as core-fucosylated PSA may be a diagnostic biomarker for the differentiation of prostate cancer and other prostatic diseases, such as benign prostatic hyperplasia (BPH). Furthermore, the described strategy could be used to monitor potential changes in site-specific core-fucosylation of other low-concentrated glycoproteins, which could serve as more specific markers ("marker refinement") in cancer research.
Assuntos
Glicoproteínas/sangue , Glicosídeo Hidrolases/metabolismo , Antígeno Prostático Específico/sangue , Cromatografia Líquida , Glicoproteínas/metabolismo , Glicosilação , Humanos , Antígeno Prostático Específico/metabolismo , Espectrometria de Massas em TandemRESUMO
BACKGROUND: High-throughput peptide and protein identification technologies have benefited tremendously from strategies based on tandem mass spectrometry (MS/MS) in combination with database searching algorithms. A major problem with existing methods lies within the significant number of false positive and false negative annotations. So far, standard algorithms for protein identification do not use the information gained from separation processes usually involved in peptide analysis, such as retention time information, which are readily available from chromatographic separation of the sample. Identification can thus be improved by comparing measured retention times to predicted retention times. Current prediction models are derived from a set of measured test analytes but they usually require large amounts of training data. RESULTS: We introduce a new kernel function which can be applied in combination with support vector machines to a wide range of computational proteomics problems. We show the performance of this new approach by applying it to the prediction of peptide adsorption/elution behavior in strong anion-exchange solid-phase extraction (SAX-SPE) and ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC). Furthermore, the predicted retention times are used to improve spectrum identifications by a p-value-based filtering approach. The approach was tested on a number of different datasets and shows excellent performance while requiring only very small training sets (about 40 peptides instead of thousands). Using the retention time predictor in our retention time filter improves the fraction of correctly identified peptide mass spectra significantly. CONCLUSION: The proposed kernel function is well-suited for the prediction of chromatographic separation in computational proteomics and requires only a limited amount of training data. The performance of this new method is demonstrated by applying it to peptide retention time prediction in IP-RP-HPLC and prediction of peptide sample fractionation in SAX-SPE. Finally, we incorporate the predicted chromatographic behavior in a p-value based filter to improve peptide identifications based on liquid chromatography-tandem mass spectrometry.
Assuntos
Biologia Computacional/métodos , Aprendizagem , Peptídeos/classificação , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Bovinos , Galinhas , Cromatografia Líquida/métodos , Cromatografia Líquida/estatística & dados numéricos , Cromatografia Líquida/tendências , Biologia Computacional/estatística & dados numéricos , Biologia Computacional/tendências , Cavalos , Humanos , Peptídeos/análise , Proteômica/estatística & dados numéricos , Proteômica/tendências , Análise de Sequência de Proteína/métodos , Análise de Sequência de Proteína/estatística & dados numéricos , Análise de Sequência de Proteína/tendências , Espectrometria de Massas em Tandem/estatística & dados numéricos , Espectrometria de Massas em Tandem/tendênciasRESUMO
The 42 amino acid form of amyloid ß (Aß1-42) in cerebrospinal fluid (CSF) has been widely accepted as a central biomarker for Alzheimer's disease. Several immunoassays for CSF Aß1-42 are commercially available, but can suffer from between laboratory and batch-to-batch variability as well as lack of standardisation across assays. As a consequence, no general cut-off values have been established for a specific context of use (e.g., clinical diagnostics) and selection of individuals for enrolment in clinical trials (patient stratification) remains challenging. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has initiated a working group for CSF proteins (WG-CSF) to facilitate standardisation of CSF Aß1-42 measurement results. The efforts of the IFCC WG-CSF include the development of certified reference materials (CRMs) and reference measurement procedures (RMPs) for key biomarkers. Two candidate RMPs for quantification of Aß1-42 in CSF based on liquid chromatography tandem mass spectrometry have been developed and tested in two ring trials. Furthermore, two commutability studies including native CSF pools, artificial CSF and spiked materials have been completed. On the basis of these studies, human CSF pools containing only endogenous Aß1-42 at three concentrations were selected as the format for future CRMs that are now being processed.
Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Testes de Química Clínica/normas , Fragmentos de Peptídeos/líquido cefalorraquidiano , Biomarcadores/líquido cefalorraquidiano , Humanos , Padrões de ReferênciaRESUMO
A fast and sensitive reference method for quantification of Δ(9) -tetrahydrocannabinol (THC) and its main metabolite 11-nor-9-carboxy-Δ(9) -tetrahydrocannabinol (THCCOOH) in oral fluid is described in this study. Samples were collected using an oral specimen collection device, followed by solid-phase extraction and liquid chromatography-tandem mass spectrometry analysis. Chromatographic separation of the analytes was achieved by gradient elution on a reversed-phase column with subsequent detection by electrospray triple quadrupole mass spectrometry in positive ionization multiple reaction monitoring mode. Quantification was performed by means of deuterated analogues of the analytes as internal standards. Total run time of the assay was 12 min. The method allowed sensitive quantification of both analytes at a limit of quantification of 0.2 ng/ml. This sensitivity is essential for analysis of samples collected with the Intercept Oral Fluid Collection device (OraSure) and an assay for simultaneous quantification of THC and THCCOOH in saliva has not yet been described. The calibration curves for THC and THCCOOH were linear in the range between 0.25 and 8 ng/ml (r(2) > 0.99). Ion suppression effects from endogenous or exogenous interferences were investigated using selected model substances (albumin, ascorbic acid, bilirubin, hemoglobin, breath spray, cigarette, chewing gum, chewing tobacco, candy, tooth whitening, and Tums antacid). These substances were chosen because of the high probability of their presence in the collected samples. None of the 11 endogenous model interferences altered the accuracy of analysis, demonstrating good robustness of the method with respect to interferences in common hygiene products, medicine, tobacco and naturally occurring endogenous substances.
Assuntos
Dronabinol/análogos & derivados , Dronabinol/análise , Alucinógenos/análise , Saliva/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Detecção do Abuso de Substâncias/métodos , Cannabis/química , Cromatografia Líquida/economia , Cromatografia Líquida/métodos , Dronabinol/isolamento & purificação , Alucinógenos/isolamento & purificação , Humanos , Sensibilidade e Especificidade , Extração em Fase Sólida/economia , Extração em Fase Sólida/métodos , Espectrometria de Massas por Ionização por Electrospray/economia , Detecção do Abuso de Substâncias/economia , Espectrometria de Massas em Tandem/economia , Espectrometria de Massas em Tandem/métodos , Fatores de TempoRESUMO
OBJECTIVES: For quantification of 25-hydroxyvitamin D(3) (25OH-D(3)), 25-hydroxyvitamin D(2) (25OH-D(2)), 3-epi-25-hydroxyvitamin D(3) (3-epi-25OH-D(3)) and 24R,25-dihydroxyvitamin D(3) (24R,25(OH)(2)-D(3)) in human serum a high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed and validated. DESIGN AND METHODS: After protein precipitation further purification is achieved with on-line sample preparation using a reversed phase (RP) C-4 column. Chromatographic separation is realized by a RP-column with core shell material and pentafluorophenyl (PFP) selectivity. Atmospheric pressure chemical ionization in the positive ion mode with multi-reaction monitoring is used for analyte detection. RESULTS: Baseline separation of the analytes is achieved below 10 min. The method is linear over the range 4.0-265.3 nmol/L for 25OH-D(3), 3.9-183.6 nmol/L for 25OH-D(2), 2.0-133.8 nmol/L for 3-epi-25OH-D(3) and 2.8-129.9 nmol/L for 24R,25(OH)(2)-D(3) (r(2)>0.998). The limit of quantification is 4.0 nmol/L for 25OH-D(3), 3.9 nmol/L for 25OH-D(2), 2.0 nmol/L for 3-epi-25OH-D(3) and 2.8 nmol/L for 24R,25(OH)(2)-D(3). The CVs for the intra-day and inter-day precision are <5% and <4%, respectively. Metabolite levels for a set of 50 human serum samples have been determined and resulted in the detection of considerable amounts of 3-epi-25OH-D(3) and 24R,25(OH)(2)-D(3). CONCLUSIONS: This highly specific HPLC-MS/MS method is suitable for vitamin D profiling. There is a correlation between 25OH-D(3) and 24R,25(OH)(2)-D(3). Serum concentration of 24R,25(OH)(2)-D(3) increases disproportionally with increasing concentration of 25OH-D(3).
Assuntos
25-Hidroxivitamina D 2/sangue , Análise Química do Sangue/normas , Hidroxicolecalciferóis/sangue , Espectrometria de Massas em Tandem/normas , 25-Hidroxivitamina D 2/isolamento & purificação , Calibragem , Cromatografia Líquida de Alta Pressão , Humanos , Hidroxicolecalciferóis/isolamento & purificação , Limite de Detecção , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
The combination of a two-dimensional peptide separation scheme based on reversed-phase and ion-pair reversed phase HPLC with a computational method to model and predict retention times in both dimensions is described. The algorithm utilizes statistical learning to establish a retention model from about 200 peptide retention times and their corresponding sequences. The application of retention time prediction to the peptides facilitated an increase in true positive peptide identifications upon lowering mass spectrometric scoring thresholds and concomitantly filtering out false positives on the basis of predicted retention times. An approximately 19% increase in the number of peptide identifications at a q-value of 0.01 was achievable in a whole proteome measurement.
Assuntos
Inteligência Artificial , Fragmentos de Peptídeos/análise , Peptídeos/análise , Proteômica/métodos , Cromatografia Líquida de Alta Pressão/métodos , Concentração de Íons de Hidrogênio , Myxococcales/química , Análise de Regressão , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodos , Fatores de TempoRESUMO
Shotgun proteome analysis of the myxobacterial model strain for secondary metabolite biosynthesis Sorangium cellulosum was performed employing off-line two-dimensional high-pH reversed-phase HPLC x low-pH ion-pair reversed-phase HPLC and dual tandem mass spectrometry with collision-induced dissociation (CID) and electron transfer dissociation (ETD) as complementary fragmentation techniques. Peptide identification using database searching was optimized for ETD fragment spectra to obtain the maximum number of identifications at equivalent false discovery rates (1.0%) in the evaluation of both fragmentation techniques. In the database search of the CID MS/MS data, the mass tolerance was set to the well-established 0.3 Da window, whereas for ETD data, it was widened to 1.1 Da to account for hydrogen-rearrangement in the radical-intermediate of the peptide precursor ion. To achieve a false discovery rate comparable to the CID results, we increased the significance threshold for peptide identification to 0.001 for the ETD data. The ETD based analysis yielded about 74% of all peptides and about 78% of all proteins compared to the CID-method. In the combined data set, 952 proteins of S. cellulosum were confidently identified by at least two peptides per protein, facilitating the study of the function of regulatory proteins in the social myxobacteria and their role in secondary metabolism.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Myxococcales/metabolismo , Mapeamento de Peptídeos/métodos , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Algoritmos , Bases de Dados de Proteínas , Armazenamento e Recuperação da InformaçãoRESUMO
Liquid chromatography coupled to mass spectrometry (LC-MS) has become a major tool for the study of biological processes. High-throughput LC-MS experiments are frequently conducted in modern laboratories, generating an enormous amount of data per day. A manual inspection is therefore no longer a feasible task. Consequently, there is a need for computational tools that can rapidly provide information about mass, elution time, and abundance of the compounds in a LC-MS sample. We present an algorithm for the detection and quantification of peptides in LC-MS data. Our approach is flexible and independent of the MS technology in use. It is based on a combination of the sweep line paradigm with a novel wavelet function tailored to detect isotopic patterns of peptides. We propose a simple voting schema to use the redundant information in consecutive scans for an accurate determination of monoisotopic masses and charge states. By explicitly modeling the instrument inaccuracy, we are also able to cope with data sets of different quality and resolution. We evaluate our technique on data from different instruments and show that we can rapidly estimate mass, centroid of retention time, and abundance of peptides in a sound algorithmic framework. Finally, we compare the performance of our method to several other techniques on three data sets of varying complexity.