Intact-Cell MALDI-ToF Mass Spectrometry for the Authentication of Drug-Adapted Cancer Cell Lines.
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ABSTRACT:
SUBMITTER: Povey JF
PROVIDER: S-EPMC6830094 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Top down proteomics in a TOF-TOF instrument was further explored by examining the fragmentation of multiply charged precursors ions generated by matrix-assisted laser desorption ionization. Evaluation of sample preparation conditions allowed selection of solvent/matrix conditions and sample deposition methods to produce sufficiently abundant doubly and triply charged precursor ions for subsequent CID experiments. As previously reported, preferential cleavage was observed at sites C-terminal to acidic residues and N-terminal to proline residues for all ions examined. An increase in nonpreferential fragmentation as well as additional low mass product ions was observed in the spectra from multiply charged precursor ions providing increased sequence coverage. This enhanced fragmentation from multiply charged precursor ions became increasingly important with increasing protein molecular weight and facilitates protein identification using database searching algorithms. The useable mass range for MALDI TOF-TOF analysis of intact proteins has been expanded to 18.2 kDa using this approach.
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Project description:Routine identification of pathogens by MALDI-TOF MS (matrix-assisted laser desorption ionisation time-of-flight mass spectrometry) is based on the fingerprint of intracellular proteins. This work evaluated the use of MALDI-TOF MS for the identification of extracellular pathogen factors. A Staphylococcus aureus isolate from a food contaminant was exponentially grown in liquid cultures. Secreted proteins were collected using methanol⁻ chloroform precipitation and analysed by MALDI-TOF MS. A main peak m/z 28,250 was demonstrated, which was identified as S.aureus enterotoxin type B (SEB) by using the pure authentic SEB reference of 28.2 kDa and by amino acid sequence analysis. SEB was also detected in this intact form following pasteurization and cooking treatments. Further application of the elaborated MALDI-TOF MS protocol resulted in the detection of SEA at m/z 27,032 and SEC at m/z 27,629. In conclusion, a simple sample preparation from S.aureus cultures and an easy-to-perform identification of pathogen factors SE in intact form represents a promising next-generation application of MALDI-TOF MS.
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Project description:Although procyanidins constitute a unique class of polymeric plant secondary metabolites with a variety of biological properties including potent antioxidant activity, structure determination has been challenging, and structures of many complex procyanidins remain uncertain. To expedite the characterization of procyanidins, negative ion matrix-assisted laser desorption ionization high-energy collision-induced dissociation tandem time-of-flight (MALDI-ToF/ToF) mass spectra of 20 isolated procyanidins containing catechin and epicatechin subunits with degrees of polymerization up to five were obtained and evaluated. Structurally significant fragmentation pathways of singly charged, deprotonated molecules were identified representing quinone methide, heterocyclic ring fission, and retro-Diels-Alder fragmentation. The interpretation of the tandem mass spectra for sequencing A-type, B-type, mixed-type, linear, and branched procyanidins is explained using specific examples of each.
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Project description:BackgroundOligosaccharidoses, which belong to the lysosomal storage diseases, are inherited metabolic disorders due to the absence or the loss of function of one of the enzymes involved in the catabolic pathway of glycoproteins and indirectly of glycosphingolipids. This enzymatic deficiency typically results in the abnormal accumulation of uncompletely degraded oligosaccharides in the urine. Since the clinical features of many of these disorders are not specific for a single enzyme deficiency, unambiguous screening is critical to limit the number of costly enzyme assays which otherwise must be performed.MethodsHere we provide evidence for the advantages of using a MALDI-TOF/TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometric (MS) method for screening oligosaccharidoses. Urine samples from previously diagnosed patients or from unaffected subjects were randomly divided into a training set and a blind testing set. Samples were directly analyzed without prior treatment.ResultsThe characteristic MS and MS/MS molecular profiles obtained allowed us to identify fucosidosis, aspartylglucosaminuria, GM1 gangliosidosis, Sandhoff disease, α-mannosidosis, sialidosis and mucolipidoses type II and III.ConclusionsThis method, which is easily run in less than 30 minutes, is performed in a single step, and is sensitive and specific. Invaluable for clinical chemistry purposes this MALDI-TOF/TOF mass spectrometry procedure is semi-automatizable and suitable for the urinary screening of oligosacharidoses.
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Project description:Because of their complexity, the separation of intact proteins from complex mixtures is an important step to comparative proteomics and the identification and characterization of the proteins by mass spectrometry (MS). In the study reported, we evaluated the use of nonporous-reversed-phase (np-RP)-HPLC for intact protein separation prior to MS analyses. The separation system was characterized and compared to 1D-SDS-PAGE electrophoresis in terms of resolution and sensitivity. We demonstrate that np-RP-HPLC protein separation is highly reproducible and provides intact protein fractions which can be directly analyzed by MALDI-TOF-MS for intact molecular weight determination. An in-well digestion protocol was developed, allowing for rapid protein identification by peptide mass fingerprinting (PMF) and resulted in comparable or improved peptide recovery compared with in-gel digestion. The np-RP sensitivity of detection by UV absorbance at 214 nm for intact proteins was at the low ng level and the sensitivity of peptide analysis by MALDI-TOF-MS was in the 10-50 fmol level. A membrane protein fraction was characterized to demonstrate application of this methodology. Among the identified proteins, multiple forms of vimentin were observed. Overall, we demonstrate that np-RP-HPLC followed by MALDI-TOF-MS allows for rapid, sensitive, and reproducible protein fractionation and very specific protein characterization by integration of PMF analysis with MS intact molecular weight information.
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Project description:The identification of proteins by tandem mass spectrometry relies on knowledge of the products produced by collision-induced dissociation of peptide ions. Most previous work has focused on fragmentation statistics for ion trap systems. We analyzed fragmentation in MALDI TOF/TOF mass spectrometry, collecting statistics using a curated set of 2459 MS/MS spectra and applying bootstrap resampling to assess confidence intervals. We calculated the frequency of 18 product ion types, the correlation between both mass and intensity with ion type, the dependence of amide bond breakage on the residues surrounding the cleavage site, and the dependence of product ion detection on residues not adjacent to the cleavage site. The most frequently observed were internal ions, followed by y ions. A strong correlation between ion type and the mass and intensity of its peak was observed, with b and y ions producing the most intense and highest mass peaks. The amino acids P, W, D, and R had a strong effect on amide bond cleavage when situated next to the breakage site, whereas residues including I, K, and H had a strong effect on product ion observation when located in the peptide but not adjacent to the cleavage site, a novel observation.
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Project description:BACKGROUND:Daratumumab, a therapeutic IgG kappa monoclonal antibody, can cause a false positive interference on electrophoretic assays that are routinely used to monitor patients with monoclonal gammopathies. In this study, we evaluate the ability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to distinguish daratumumab from disease-related IgG kappa monoclonal proteins (M-protein). METHODS:Waste clinical samples from 31 patients who were receiving daratumumab and had a history of IgG kappa monoclonal gammopathy were collected. Immunoglobulins were purified from serum and analyzed by MALDI-TOF MS. Mass spectra were assessed for the presence of distinct monoclonal proteins. For samples in which only one monoclonal peak was identified near the expected m/z of daratumumab, the Hydrashift 2/4 Daratumumab Assay was used to confirm the presence of an M-protein. RESULTS:Using MALDI-TOF MS, daratumumab could be distinguished from M-proteins in 26 out of 31 samples (84%). Results from 2 samples were inconclusive since the M-protein was not detected by the Hydrashift assay and may also be undetectable by MALDI-TOF MS. Comparatively, daratumumab was distinguishable from M-proteins in 14 out of 31 samples (45%) by immunofixation. CONCLUSIONS:MALDI-TOF MS offers greater specificity compared to immunofixation for distinguishing daratumumab from M-proteins.
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Project description:A method of analysis is presented that utilizes matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to monitor the kinetics and products of RNA cleavage, by use of a program designed to mass-match observed MS peaks with predicted RNA cleavage products. The method is illustrated through application to the study of targeted oxidation of RNA stem loops from HIV-1 Rev Response Element mRNA (RRE RNA) and ribosomal 16S A-site RNA (16S RNA) by metallonucleases. Following incubation of each RNA with catalysts and/or redox co-reactants, reaction mixtures were desalted, and MALDI-TOF MS was used to monitor both time-resolved formation of cleavage products and disappearance of full-length RNA. For each RNA, a unique list was generated that contained the predicted masses of both the full-length, and all of the possible RNA cleavage fragments that resulted from the combination of all possible cleavage sites and each of the six expected overhangs formed at nascent termini adjacent to the cleavage sites. The overhangs corresponded to 2',3'-cyclic phosphate, 3'-phosphate, 3'-phosphoglycolate, 5'- hydroxyl and 5'- phosphate, which corresponded to differing oxidative, hydrolytic, and/or 2'-OH-mediated-endonucleolytic modes of scission. Each mass spectrum was compared with a corresponding list of predicted masses, and peaks were rapidly assigned by use of a Perl script, with a mass-matching tolerance of 200 ppm. Both time-dependent cleavage mediated by metallonucleases and MALDI-TOF-induced fragmentation were observed, and these were distinguished by time-dependent experiments. The resulting data allowed a semi-quantitative assessment of the rate of formation of each overhang at each nucleotide position. Limitations included artifactual skewing of quantification by mass bias, a limited mass range for quantification, and a lack of detection of secondary cleavage products. Nevertheless, the method presented herein provides a rapid, accurate, highly-detailed and semi-quantitative analysis of RNA cleavage that should be widely applicable.
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Project description:Dermatophytoses represent a major health burden in animals and man. Zoophilic dermatophytes usually show a high specificity to their original animal host but a zoonotic transmission is increasingly recorded. In humans, these infections elicit highly inflammatory skin lesions requiring prolonged therapy even in the immunocompetent patient. The correct identification of the causative agent is often crucial to initiate a targeted and effective therapy. To that end, matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) represents a promising tool. The objective of this study was to evaluate the reliability of species identification of zoophilic dermatophytes using MALDI-TOF MS. The investigation of isolates from veterinary clinical samples suspicious of dermatophytoses suggests a good MALDI-TOF MS based identification of the most common zoophilic dermatophyte Microsporum canis. Trichophyton (T.) spp. usually achieved scores only around the cutoff value for secure species identification because of a small number of reference spectra. Moreover, these results need to be interpreted with caution due to the close taxonomic relationship of dermatophytes being reflected in very similar spectra. In our study, the analysis of 50 clinical samples of hedgehogs revealed no correct identification using the provided databases, nor for zoophilic neither for geophilic causative agents. After DNA sequencing, adaptation of sample processing and an individual extension of the in-house database, acceptable identification scores were achieved (T. erinacei and Arthroderma spp., respectively). A score-oriented distance dendrogram revealed clustering of geophilic isolates of four different species of the genus Arthroderma and underlined the close relationship of the important zoophilic agents T. erinacei, T. verrucosum and T. benhamiae by forming a subclade within a larger cluster including different dermatophytes. Taken together, MALDI-TOF MS proofed suitable for the identification of zoophilic dermatophytes provided fresh cultures are used and the reference library was previously extended with spectra of laboratory-relevant species. Performing independent molecular methods, such as sequencing, is strongly recommended to substantiate the findings from morphologic and MALDI-TOF MS analyses, especially for uncommon causative agents.
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