共查询到20条相似文献,搜索用时 15 毫秒
1.
Heudi O Barteau S Zimmer D Schmidt J Bill K Lehmann N Bauer C Kretz O 《Analytical chemistry》2008,80(11):4200-4207
Although LC-MS methods are increasingly used for the absolute quantification of proteins, the lack of appropriate internal standard (IS) hinders the development of rapid and standardized analytical methods for both in vitro and in vivo studies. Here, we have developed a novel method for the absolute quantification of a therapeutic protein, which is monoclonal antibody (mAb). The method combines liquid chromatography tandem mass spectrometry (LC-MS/MS) and protein cleavage isotope dilution mass spectrometry with the isotope-labeled mAb as IS. The latter was identical to the analyzed mAb with the exception that each threonine contains four (13)C atoms and one (15)N atom. Serum samples were spiked with IS prior to the overnight trypsin digestion and subsequent sample cleanup. Sample extracts were analyzed on a C18 ACE column (150 mm x 4.6 mm) using an LC gradient time of 11 min. Endogenous mAb concentrations were determined by calculating the peak height ratio of its signature peptide to the corresponding isotope-labeled peptide. The linear dynamic range was established between 5.00 and 1000 microg/mL mAb with accuracy and precision within +/-15% at all concentrations and below +/-20% at the LLOQ (lower limit of quantification). The overall method recovery in terms of mAb was 14%. The losses due to sample preparation (digestion and purification) were 72% from which about 32% was due to the first step of the method, the sample digestion. This huge loss during sample preparation strongly emphasizes the necessity to employ an IS right from the beginning. Our method was successfully applied to the mAb quantification in marmoset serum study samples, and the precision obtained on duplicate samples was, in most cases, below 20%. The comparison with enzyme-linked immunosorbent assay (ELISA) showed higher exposure in terms of AUC and Cmax with the LC-MS/MS method. Possible reasons for this discrepancy are discussed in this study. The results of this study indicate that our LC-MS/MS method is a simple, rapid, and precise approach for the therapeutic mAb quantification to support preclinical and clinical studies. 相似文献
2.
The stoichiometry of protein phosphorylation significantly impacts protein function. The development of quantitative techniques in mass spectrometry has generated the ability to systematically monitor the regulation levels of various proteins. This study reports an integrated methodology using cerium oxide nanoparticles and isobaric tandem mass tag (TMT) labeling to assess absolute stoichiometries of protein phosphorylation. This protocol was designed to directly measure the dephosphorylation levels for a known phosphorylation site, therefore allowing for quantification of phosphosites. Both the accuracy and precision of the method were verified using standard peptides and protein tryptic digests. This novel method was then applied to quantify phosphorylations on eukaryotic initiation factor 3H (eIF3H), a protein integral to overall eukaryotic protein translation initiation. To date, this is the first report of assessment of protein phosphorylation quantification on eIF3. 相似文献
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An isotope dilution mass spectrometric (ID/MS) method for cholesterol is described that uses capillary gas chromatography with cholesterol-13C3 as the labeled internal standard. Labeled and unlabeled cholesterol are converted to the trimethylsilyl ether. Combined capillary column gas chromatography and electron impact mass spectrometry are used to obtain the abundance ratio of the unlabeled and labeled [M+.] ions from the derivative. Quantitation is achieved by measurement of each sample between measurements of two standards whose unlabeled/labeled ratios bracket that of the sample. Seven pools were analyzed by this method: standard reference material (SRM) 1951, which consists of three frozen serum pools with low, medium, and high levels of cholesterol; SRM 1952, which consists of three freeze-dried serum pools with low, medium, and high levels of cholesterol; and SRM 909, a freeze-dried serum pool. The method is a modification of our original definitive method for cholesterol. The modified method uses much better chromatographic separations to assure specificity and a new method of implementing selected ion monitoring on a magnetic mass spectrometer to obtain high-precision measurements of ion intensity ratios on narrow gas chromatographic peaks. The modified method has a coefficient of variation (CV) of 0.22%, which is an improvement over the original method's CV of 0.36%. The measurements were found to be free of interference. The high precision and absence of bias qualify this method as a candidate definitive method. 相似文献
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D G Patterson L Hampton C R Lapeza W T Belser V Green L Alexander L L Needham 《Analytical chemistry》1987,59(15):2000-2005
7.
Boyer AE Quinn CP Woolfitt AR Pirkle JL McWilliams LG Stamey KL Bagarozzi DA Hart JC Barr JR 《Analytical chemistry》2007,79(22):8463-8470
The lethal toxin produced during Bacillus anthracis infection is a complex of protective antigen, which localizes the toxin to the cell receptor, and lethal factor (LF), a zinc-dependent endoproteinase whose known targets include five members of the mitogen-activated protein kinase kinase (MAPKK) family of response regulators. We have developed a method for detecting functional LF in serum. Anti-LF murine monoclonal antibodies immobilized on magnetic protein G beads were used to capture and concentrate the LF from serum. The captured LF was exposed to an optimized MAPKK-based peptide substrate, which it hydrolyzed into two smaller peptides. The LF cleavage products were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and quantified by isotope dilution-MS. The entire analytical method can be performed in less than 4 h with detection of LF levels as low as 0.05 ng/mL. The method was used to quantify LF levels in serum from rhesus macaques infected with B. anthracis. Serum samples obtained at day 2 postinfection contained 30-250 ng/mL LF and illustrated the clear potential to detect LF earlier in the infection cycle. This method represents a highly specific and rapid diagnostic tool for early anthrax and has a potential additional role as a research tool for understanding toxemia and effects of medical countermeasures for anthrax. 相似文献
8.
Russell JD Hilger RT Ladror DT Tervo MA Scalf M Shortreed MR Coon JJ Smith LM 《Analytical chemistry》2011,83(6):2187-2193
Direct mass spectrometric quantification of peptides and proteins is compromised by the wide variabilities in ionization efficiency which are hallmarks of both the MALDI and ESI ionization techniques. We describe here the implementation of a fluorescence detection system for measurement of the UV-excited intrinsic fluorescence (UV-IF) from peptides and proteins just prior to their exit and electrospray ionization from an ESI capillary. The fluorescence signal provides a quantifiable measure of the amount of protein or peptide present, while direct or tandem mass spectrometric analysis (MS/MS) on the ESI-generated ions provides information on identity. We fabricated an inexpensive, modular fluorescence excitation and detection device utilizing an ultraviolet light-emitting diode for excitation in a ~300 nL fluorescence detection cell integrated into the fused-silica separation column. The fluorescence signal is linear over 3 orders of magnitude with on-column limits of detection in the low femtomole range. Chromatographically separated intact proteins analyzed using UV-IF prior to top-down mass spectrometry demonstrated sensitive detection of proteins as large as 77 kDa. 相似文献
9.
A unified extraction and quantification procedure based on stable isotope-dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed for the simultaneous determination of total homocysteine and folate (5-methyltetrahydrofolic acid and folic acid) levels in human serum and plasma. This is the first report documenting the simultaneous extraction and quantification of these structurally dissimilar analytes. Analytes are quantitatively isolated from samples (500 microL) prior to LC/MS/MS analysis using a two-step stabilization process combined with C18 solid-phase extraction. The method exhibits excellent linearity over 4 orders of magnitude for each analyte. Measurement repeatability (RSD, N = 2) ranged from 0.3% to 3% for all analytes over 1 day of analysis. Total method variability (RSD, N = 6) ranged from 0.7% to 10% for all analytes over three independent days of analysis. The accuracy and practical applicability of the method were demonstrated by applying the method to the quantitative determination of each analyte in a new NIST serum Standard Reference Material (NIST SRM 1955 Homocysteine and Folate in Frozen Human Serum) and in a small subset of normal donor plasma samples. 相似文献
10.
West-Nielsen M Høgdall EV Marchiori E Høgdall CK Schou C Heegaard NH 《Analytical chemistry》2005,77(16):5114-5123
Proteomic investigations of sera are potentially of value for diagnosis, prognosis, choice of therapy, and disease activity assessment by virtue of discovering new biomarkers and biomarker patterns. Much debate focuses on the biological relevance and the need for identification of such biomarkers while less effort has been invested in devising standard procedures for sample preparation and storage in relation to model building based on complex sets of mass spectrometric (MS) data. Thus, development of standardized methods for collection and storage of patient samples together with standards for transportation and handling of samples are needed. This requires knowledge about how sample processing affects MS-based proteome analyses and thereby how nonbiological biased classification errors are avoided. In this study, we characterize the effects of sample handling, including clotting conditions, storage temperature, storage time, and freeze/thaw cycles, on MS-based proteomics of human serum by using principal components analysis, support vector machine learning, and clustering methods based on genetic algorithms as class modeling and prediction methods. Using spiking to artificially create differentiable sample groups, this integrated approach yields data that--even when working with sample groups that differ more than may be expected in biological studies--clearly demonstrate the need for comparable sampling conditions for samples used for modeling and for the samples that are going into the test set group. Also, the study emphasizes the difference between class prediction and class comparison studies as well as the advantages and disadvantages of different modeling methods. 相似文献
11.
D K Bryant R C Orlando C Fenselau R C Sowder L E Henderson 《Analytical chemistry》1991,63(11):1110-1114
A number of phosphatidylcholines have been isolated from an HIV-1/MN preparation by reversed-phase high-performance liquid chromatography (HPLC) and analyzed by fast atom bombardment mass spectrometry (FABMS), FABMS/MS, and FABMS/MS/MS in both positive- and negative-ion modes. Negative-ion FABMS/MS with high-energy collisions was used to identify the length of the acyl groups and the degree of saturation, as well as their position on the glyceride group. FABMS/MS in the positive-ion mode was used to identify the polar head group. Negative-ion FABMS/MS/MS was used to locate positions of double bonds in acyl groups. We find that four-sector tandem mass spectrometry with high-energy collisional activation provides qualitative analysis of viral phosphatidyl lipids in considerable detail, as well as semiquantitative information. Approximate quantitation of the phosphatidylcholine content of the HIV-1/MN preparation by measuring relative peak heights of molecular ions in FABMS reveals an array of phosphatidylcholines consistent with that found in human erythrocytes, indicating the likely source of lipids in the viral membrane to be the host cell membrane. 相似文献
12.
Picomole amounts of endogenous methionine-enkephalin (ME = YGGFM) were quantified in 11 individual human pituitaries by fast atom bombardment mass spectrometry methods. Quantification was based either upon the comparison of the molecular ion (MH+) current of endogenous ME versus the current of a deuterated ME internal standard (d5-ME) or, similarly, upon the unimolecular decomposition MH+----YGGF-+ In the first field-free region to produce the unique tetrapeptide fragment ion. The latter method used the multiple reaction monitoring (MRM) mode. Native ME was purified with an octadecylsilyl (ODS) disposable cartridge and with multidimensional reversed-phase high-performance liquid chromatography. The amounts of ME determined were 18.26 +/- 19.98 ng of ME/mg of protein with the MH+ method and 15.28 +/- 16.59 ng of ME/mg of protein with the MRM method. A fraction (ca. 4%) of the total amount of ME from one pituitary was used to acquire these quantitative data, and ca. half of the remaining amount of a separate sample (no d5-ME added) was used to obtain a linked scan at constant B/E (B, magnetic field; E, electric field) of the ME MH+ at 574 u to produce the amino acid sequence determining fragment ions at m/z 297, 354, 411, 397, 278, and 425 u corresponding to Y2", Y3", Y4", A4, B3, and B4, respectively. That product ion spectrum was similar to a scan of 100 ng of synthetic ME. We calculated that the amount of pentapeptide for the MRM experiments corresponded to a total of 30 ng (52 pmol) of ME on the probe tip during quantification. On the other hand, we estimated that 3 times more, or 90 ng (156 pmol), ME was on the probe tip during acquisition of the product ion spectrum. 相似文献
13.
Frontal immunoaffinity chromatography with mass spectrometric detection: a method for finding active compounds from traditional Chinese herbs 总被引:2,自引:0,他引:2
Frontal affinity chromatography (FAC) using immobilized polyclone antibodies of compound A coupled with mass spectrometry was used for the screening of affinity compounds from an extract of Phyllanthus urinaria L. Mass spectrometry was used as an analyzer of FAC. It can analyze the frontal affinity chromatogram of each compound of the extract in one program. The extract was dissolved in 2 mM NH4OAc at a concentration of 10 microg/ mL, then loaded on the immobilized antibody column, and data were collected from mass spectrometry to get a frontal affinity chromatogram. The screening of extract resulted in brevifolin, brevifolin carboxylic acid, corilagin, ellagic acid, and phyllanthusiin U. Activity analyses give high inhibitory activities to these compounds. This research work afforded us a new approach to find new leading compounds from nature or a man-made combinatorial library that have different structure styles or to find substitutes for the synthetic active compound that has high toxicity. 相似文献
14.
Hemoglobin-based oxygen therapeutics are prepared by reaction of hemoglobin with cross-linking molecules and are utilized as blood substitutes. They can be used as doping agents to increase the oxygen-carrying capacity of hemoglobin. We have compared a glutaraldehyde-polymerized bovine hemoglobin (Oxyglobin, Biopure Corp.) with natural bovine hemoglobin by mass spectrometry in order to detect specific fragment ions of the cross-linked protein for further potential applications in doping control of human blood samples. HCl acid (6 N) hydrolysis was performed in parallel on both proteins. Hydrolysates were then analyzed by direct infusion electrospray mass spectrometry (ESIMS) using a triple quadrupole mass spectrometer. Confirmation and precision were obtained by LC-ESIMS(n) experiments performed on an ion trap mass spectrometer. Chromatographic and mass spectrometry data allowed detection of two potential Oxyglobin-specific ions--m/z 299 and 399--that were shown to lose a 159 u neutral fragment under collision-induced dissociation conditions. Thus, monitoring of constant neutral loss of 159 u on acid hydrolysates of human serum samples spiked with different amounts of Oxyglobin has proved to be an efficient screening method to specifically detect and identify Oxyglobin. LC-MS of the spiked serum sample hydrolysates enabled detection of Oxyglobin at a detection limit of 4 g x L(-1). 相似文献
15.
Minerva L Boonen K Menschaert G Landuyt B Baggerman G Arckens L 《Analytical chemistry》2011,83(20):7682-7691
MALDI mass spectrometry imaging (MSI) is a promising technique in the field of molecular (immuno)histology but is confronted with the problematic large-scale identification of peptides from thin tissue sections. In this study we present a workflow that significantly increased the number of identified peptides in a given MALDI-MSI data set and we evaluated its power concerning relative peptide quantifications. Fourier transform mass spectrometry (FTMS) profiling on matrix-coated thin tissue sections allowed us to align spectra of different MS sources, matching identical peaks in the process, thus linking MSI data to tandem mass spectrometry (MS/MS) on one hand and semiquantitative liquid chromatography (LC)/MS data on the other. Bonanza clustering was applied in order to group MS/MS spectra of structurally related peptides, making it possible to infer the identity of MSI-detected compounds based on identified members within the same cluster, effectively increasing the number of identifications in a single MSI data set. Out of 136 detected peptides with MALDI-MSI, we were able to identify 46 peptides. For 31 of these, a LC/quadrupole time-of-flight (QTOF) counterpart was detected, and we observed similar obese (ob/ob) to wild-type (wt) peak intensity ratios for 18 peptides. This workflow significantly increased the number of identifications of peptide masses detected with MALDI-MSI and evaluated the power of this imaging method for relative quantification of peptide levels between experimental conditions. 相似文献
16.
Deterding LJ Bhattacharjee S Ramirez DC Mason RP Tomer KB 《Analytical chemistry》2007,79(16):6236-6248
In an effort to determine the utility of top-down mass spectrometric methodologies for the characterization of protein radical adducts, top-down approaches were investigated and compared to the traditional bottom-up approaches. Specifically, the nature of the radicals on human myoglobin induced by the addition of hydrogen peroxide and captured by the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was investigated. The most abundant ion observed in the electrospray mass spectrum of this reaction mixture corresponds in mass to the human myoglobin plus one DMPO molecule. In addition, a second ion of lower abundance is observed, which corresponds to a second DMPO molecule being trapped on myoglobin. Top-down analyses using Fourier transform ion cyclotron resonance mass spectrometry can be used to characterize proteins and, thus, were performed on several different charge-state ions of both the native and the mono-DMPO nitrone adduct of human myoglobin. Data produced from the top-down analyses are very complex yet information rich. In the case of DMPO-modified human myoglobin, the top-down data localized the DMPO spin trap to residues 97-110 of the myoglobin. The observation of the y43+5 fragment ion arising from C-terminal cleavage to the cysteine-110 residue in the MS/MS spectrum of DMPO-modified myoglobin and not in the unmodified myoglobin implicates a change to this residue, specifically, DMPO adduction. On the other hand, using the traditional bottom-up approach of peptide mapping and MS sequencing methodologies, two DMPO radical adducts on human myoglobin were identified, Cys-110 and Tyr-103. The bottom-up approach is more proven and robust than the top-down methodologies. Nonetheless, the bottom-up and top-down approaches to protein characterization are complementary rather than competitive approaches with each having its own utility. 相似文献
17.
C Jiménez-Castells S Defaus A Moise M Przbylski D Andreu R Gutiérrez-Gallego 《Analytical chemistry》2012,84(15):6515-6520
Interest in powerful, nanosized tools to analyze in detail glycan-protein interactions has increased significantly over recent years. Here, we report two complementary approaches to characterize such interactions with high sensitivity, low sample consumption, and without the need for sample labeling, namely, surface plasmon resonance (SPR) and an approach that combines limited proteolysis and mass spectrometry. Combination of these two approaches to investigate glycan-protein interactions allows (1) to characterize interactions through kinetic and thermodynamic parameters, (2) to capture efficiently the carbohydrate-binding protein, and (3) to identify the interacted protein and its carbohydrate binding site by mass spectrometry. As a proof of principle, the interaction of the galactose-specific legume lectin Erythrina cristagalli agglutinin with several sugars has been characterized in-depth by means of these two approaches. 相似文献
18.
Mortier KA Renard V Verstraete AG Van Gussem A Van Belle S Lambert WE 《Analytical chemistry》2005,77(14):4677-4683
A quantitative method for the simultaneous determination of docetaxel (Taxotere), paclitaxel (Taxol), 6alpha-hydroxypaclitaxel, and p-3'-hydroxypaclitaxel in human plasma and oral fluid is developed and validated. Oral fluid (this term is now preferred to saliva) was sampled with a Salivette collection device. The procedure used a simple liquid/liquid extraction with methyl tert-butyl ether followed by LC-ESI-MS/MS. Gradient elution was applied and provided increased robustness to ion suppression by the drug formulation vehicle (polysorbate 80 and Cremophor EL). Adduct ion formation with sodium and potassium was noticed and controlled by mobile-phase optimization. The protonated analytes generated in the positive ion mode were monitored through multiple reaction monitoring. Calibration was performed by internal standardization with cephalomannine, and regression curves were constructed ranging between 2 and 1000 ng/mL in plasma and 0.125 and 62.5 ng/mL in oral fluid, using a weighing factor of 1/x2. The regression curves were quadratic for paclitaxel and docetaxel and linear for the paclitaxel metabolites. Accuracy varied from 91.3 to 103.6%, and imprecision did not exceed 12.7% for all analytes in plasma and oral fluid. In conclusion, a sensitive and robust method was obtained, which fulfilled all validation criteria. 相似文献
19.
Lippok S Seidel SA Duhr S Uhland K Holthoff HP Jenne D Braun D 《Analytical chemistry》2012,84(8):3523-3530
The direct quantification of both the binding affinity and absolute concentration of disease-related biomarkers in biological fluids is particularly beneficial for differential diagnosis and therapy monitoring. Here, we extend microscale thermophoresis to target immunological questions. Optically generated thermal gradients were used to deplete fluorescently marked antigens in 2- and 10-fold-diluted human serum. We devised and validated an autocompetitive strategy to independently fit the concentration and dissociation constant of autoimmune antibodies against the cardiac β1-adrenergic receptor related to dilated cardiomyopathy. As an artificial antigen, the peptide COR1 was designed to mimic the second extracellular receptor loop. Thermophoresis resolved antibody concentrations from 2 to 200 nM and measured the dissociation constant as 75 nM. The approach quantifies antibody binding in its native serum environment within microliter volumes and without any surface attachments. The simplicity of the mix and probe protocol minimizes systematic errors, making thermophoresis a promising detection method for personalized medicine. 相似文献
20.
N. A. Gribchenkova A. V. Steblevskii E. N. Kolosov A. S. Alikhanian G. D. Nipan 《Inorganic Materials》2007,43(1):78-84
The vapor composition in the Bi-Sb-O system has been determined by Knudsen cell mass spectrometry. We have calculated the individual mass spectra of the Bi3SbO6, Bi2Sb2O6, and BiSb3O6 molecules and determined the partial pressures of all the vapor species identified. Bi3SbO7 and BiSbO4 have been shown to sublime congruently. The results have been used to construct p-x sections for the pseudobinary oxide systems Bi2O3-BiSbO4 and BiSbO4-Sb2O4. 相似文献