Intact Cell MALDI-TOF Mass Spectrometry Research Articles

Intact cell mass spectrometry coupled with machine learning reveals minute changes induced by single gene silencing

Intact (whole) cell MALDI TOF mass spectrometry is a commonly used tool in clinical microbiology for several decades. Recently it was introduced to analysis of eukaryotic cells, including cancer and stem cells. Besides targeted metabolomic and proteomic applications, the intact cell MALDI TOF mass spectrometry provides a sufficient sensitivity and specificity to discriminate cell types, isogenous cell lines or even the metabolic states. This makes the intact cell MALDI TOF mass spectrometry a promising tool for quality control in advanced cell cultures with a potential to reveal batch-to-batch variation, aberrant clones, or unwanted shifts in cell phenotype. However, cellular alterations induced by change in expression of a single gene has not been addressed by intact cell mass spectrometry yet. In this work we used a well-characterized human ovarian cancer cell line SKOV3 with silenced expression of a tumor suppressor candidate 3 gene (TUSC3). TUSC3 is involved in co-translational N-glycosylation of proteins with well-known global impact on cell phenotype. Altogether, this experimental design represents a highly suitable model for optimization of intact cell mass spectrometry and analysis of spectral data. Here we investigated five machine learning algorithms (k-nearest neighbors, decision tree, random forest, partial least squares discrimination, and artificial neural network) and optimized their performance either in pure populations or in two-component mixtures composed of cells with normal or silenced expression of TUSC3. All five algorithms reached accuracy over 90 % and were able to reveal even subtle changes in mass spectra corresponding to alterations of TUSC3 expression. In summary, we demonstrate that spectral fingerprints generated by intact cell MALDI-TOF mass spectrometry coupled to a machine learning classifier can reveal minute changes induced by alteration of a single gene, and therefore contribute to the portfolio of quality control applications in routine cell and tissue cultures.

Intact cell MALDI-TOF mass spectrometry, a promising proteomic profiling method in farm animal clinical and reproduction research

The objective of this review is to provide new insights into the possible use of a proteomic method known as Intact Cell Matrix-Assisted Laser Desorption-ionization Time-Of-Flight Mass Spectrometry (ICM-MS) in animal clinical research. Here, we give an overview of the basics of this technique, its advantages and disadvantages compared with other proteomic approaches, past applications and future perspectives. A special emphasis on its implementation in animal reproduction science is given, including examples of the reliable use of ICM-MS on fertility screening. In mammals, the ICM-MS profiles from pig epididymal spermatozoa reflect the proteome changes that they undergo during epididymal maturation and could be associated with the acquisition of fertilizing ability. In chicken, using adequate pre-processing and bioinformatics analysis tools, sperm ICM-MS profiles showed characteristic spectral features that allowed their classification according to their actual fertilizing ability. The association of ICM-MS and Top-down proteomic strategies allowed the identification of chicken fertility biomarkers candidates such as protein vitelline membrane outer layer protein 1 (VMO-1) and avian beta-defensin 10 (AvBD10). In female reproduction, a similar approach on ovarian follicular cells allowed the identification of specific markers of oocyte maturation in the oocyte and surrounding cumulus cells. Altogether, these results indicate that ICM-MS profiling could be a suitable approach for molecular phenotyping of male and female gametes.

Intact-Cell MALDI-ToF Mass Spectrometry for the Authentication of Drug-Adapted Cancer Cell Lines.

The use of cell lines in research can be affected by cell line misidentification. Short tandem repeat (STR) analysis is an effective method, and the gold standard, for the identification of the genetic origin of a cell line, but methods that allow the discrimination between cell lines of the same genetic origin are lacking. Here, we use intact cell MALDI-ToF mass spectrometry analysis, routinely used for the identification of bacteria in clinical diagnostic procedures, for the authentication of a set of cell lines consisting of three parental neuroblastoma cell lines (IMR-5, IMR-32 and UKF-NB-3) and eleven drug-adapted sublines. Principal component analysis (PCA) of intact-cell MALDI-ToF mass spectrometry data revealed clear differences between most, but not all, of the investigated cell lines. Mass spectrometry whole-cell fingerprints enabled the separation of IMR-32 and its clonal subline IMR-5. Sublines that had been adapted to closely related drugs, for example, the cisplatin- and oxaliplatin-resistant UKF-NB-3 sublines and the vincristine- and vinblastine-adapted IMR-5 sublines, also displayed clearly distinctive patterns. In conclusion, intact whole-cell MALDI-ToF mass spectrometry has the potential to be further developed into an authentication method for mammalian cells of a common genetic origin.

Intraoviductal concentrations of steroid hormones during in vitro culture changed phospholipid profiles and cryotolerance of bovine embryos.

The objective of this study was to evaluate the effect of progesterone (P4), estradiol (E2), and cortisol (CO) at intraoviductal concentrations on bovine embryo development and quality in vitro. After fertilization of in vitro matured oocytes, zygotes were cultured for 8 days in synthetic oviductal fluid, supplemented with 55 ng/ml P4, 120 pg/ml E2, 40 ng/ml CO, or their combination (ALL). Control embryos were cultured with vehicle (0.1% ethanol). Exposure to steroids did not affect the embryo developmental rate nor the mean number of cells per blastocyst. However, at 24 hr after vitrification-warming, exposure to P4 improved the proportion of embryos that re-expanded and were viable while exposure to CO decreased the proportion of viable embryos. By intact cell MALDI-TOF mass spectrometry, a total of 242 phospholipid masses of 400-1000 m/z were detected from individual fresh blastocysts. Exposure to ALL induced the highest and most specific changes in embryo phospholipids, followed by P4, E2, and CO. In particular, the m/z 546.3 and 546.4 attributed to lysophosphatidylcholines were found less abundant after exposure to P4. In conclusion, exposure of bovine embryos to intraoviductal concentrations of steroid hormones did not affect in vitro development but changed blastocyst quality in terms of cryotolerance and phospholipid profiles.

At-line Monitoring von Zellkulturprozessen mittels Massenspektrometrie

Perfusion becomes a reasonable alternative to traditional batch or fed-batch processes, partly due to the increasing availability of suitable re-usable or single-use equipment. High cell densities and long duration of perfusion processes require reliable process control. Amongst others, monitoring of cell stress becomes important. Intact cell MALDI-TOF mass spectrometry (ICM-MS) biotyping is described here as a robust at-line monitoring method of cell stress in perfusion cell culture.

Intact cell MALDI-TOF mass spectrometry on single bovine oocyte and follicular cells combined with top-down proteomics: A novel approach to characterise markers of oocyte maturation

Intact cell MALDI-TOF mass spectrometry (ICM-MS) was adapted to bovine follicular cells from individual ovarian follicles to obtain the protein/peptide signatures (<17kDa) of single oocytes, cumulus cells (CC) and granulosa cells (GC), which shared a total of 439 peaks. By comparing the ICM-MS profiles of single oocytes and CC before and after in vitro maturation (IVM), 71 different peaks were characterised, and their relative abundance was found to vary depending on the stage of oocyte meiotic maturation. To identify these endogenous biomolecules, top-down workflow using high resolution MS/MS (TD HR-MS) was performed on the protein extracts from oocytes, CC and GC. The TD HR-MS proteomic approach allowed for: (1) identification of 386 peptide/proteoforms encoded by 194 genes; and (2) characterisation of proteolysis products likely resulting from the action of kallikreins and caspases. In total, 136 peaks observed by ICM-MS were annotated by TD HR-MS (ProteomeXchange PXD004892). Among these, 16 markers of maturation were identified, including IGF2 binding protein 3 and hemoglobin B in the oocyte, thymosins beta-4 and beta-10, histone H2B and ubiquitin in CC. The combination of ICM-MS and TD HR-MS proved to be a suitable strategy to identify non-invasive markers of oocyte quality using limited biological samples. Biological significanceIntact cell MALDI-TOF mass spectrometry on single oocytes and their surrounding cumulus cells, coupled to an optimised top-down HR-MS proteomic approach on ovarian follicular cells, was used to identify specific markers of oocyte meiotic maturation represented by whole low molecular weight proteins or products of degradation by specific proteases.

MALDI-TOF mass spectrometry biotyping: At line monitoring of recombinant CHO cell cultures

Background The cultivation of mammalian cells, especially Chinese Hamster Ovary (CHO), has found widespread acceptance for the production of biopharmaceuticals. Process monitoring enforced by the PAT initiative of the FDA underwent major changes from examining the bioreactor/culture as a “black-box” towards integrated methods, helping to gain deeper process understanding by assessing the cells themselves. Previously, we presented first results of CHO batch cultures assessed in order to monitor cell stress and early apoptosis by Intact Cell MALDI-TOF Mass Spectrometry (ICM MS) biotyping, a method originally used in clinical microbiology [1]. Now, we broadened its application and addressed additional CHO cell lines (DXB11 and CHOK1), different cultivation-formats (uncontrolled Erlenmeyer flasks vs. lab scale bioreactors) and operation modes (Batch vs. Perfusion). Furthermore, we transferred the method to a compact, “low-priced” mass spectrometer (MicroFlex, Bruker Daltonik, Bremen, Germany). The experimental aim was to establish characteristic MS patterns indicative for different apoptotic states with emphasis on detection of early apoptosis ubiquitously applicable independent of CHOsubline, cultivation-format, -mode or instrumental equipment (MS).

Rapid high-throughput characterisation, classification and selection of recombinant mammalian cell line phenotypes using intact cell MALDI-ToF mass spectrometry fingerprinting and PLS-DA modelling

Despite many advances in the generation of high producing recombinant mammalian cell lines over the last few decades, cell line selection and development is often slowed by the inability to predict a cell line's phenotypic characteristics (e.g. growth or recombinant protein productivity) at larger scale (large volume bioreactors) using data from early cell line construction at small culture scale. Here we describe the development of an intact cell MALDI-ToF mass spectrometry fingerprinting method for mammalian cells early in the cell line construction process whereby the resulting mass spectrometry data are used to predict the phenotype of mammalian cell lines at larger culture scale using a Partial Least Squares Discriminant Analysis (PLS-DA) model. Using MALDI-ToF mass spectrometry, a library of mass spectrometry fingerprints was generated for individual cell lines at the 96 deep well plate stage of cell line development. The growth and productivity of these cell lines were evaluated in a 10L bioreactor model of Lonza's large-scale (up to 20,000L) fed-batch cell culture processes. Using the mass spectrometry information at the 96 deep well plate stage and phenotype information at the 10L bioreactor scale a PLS-DA model was developed to predict the productivity of unknown cell lines at the 10L scale based upon their MALDI-ToF fingerprint at the 96 deep well plate scale. This approach provides the basis for the very early prediction of cell lines’ performance in cGMP manufacturing-scale bioreactors and the foundation for methods and models for predicting other mammalian cell phenotypes from rapid, intact-cell mass spectrometry based measurements.

Identification of Lactobacillus strains of goose origin using MALDI-TOF mass spectrometry and 16S–23S rDNA intergenic spacer PCR analysis

The objective of our study was to identify Lactobacillus sp. strains of goose origin using MALDI-TOF mass spectrometry, ITS-PCR and ITS-PCR/RFLP. All three techniques proved to be valuable tools for identification of avian lactobacilli and produced comparable classification results. Lactobacillus strains were isolated from 100% of geese aged 3 weeks to 4 years, but from only 25% of chicks aged 1–10 days. Among the 104 strains isolated, we distinguished 14 Lactobacillus species. The dominant species was Lactobacillus salivarius (35.6%), followed by Lactobacillus johnsonii (18.3%), Lactobacillus ingluviei (11.5%) and Lactobacillus agilis (7.7%). The intact-cell MALDI-TOF mass spectrometry enabled rapid species identification of the lactobacilli with minimal pretreatment. However, it produced more than one identification result for 11.5% examined strains (mainly of the species L. johnsonii). ITS-PCR distinguished 12 genotypes among the isolates, but was not able to differentiate closely related strains, i.e. between Lactobacillus amylovorus and Lactobacillus kitasatonis and between Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus zeae. These species were differentiated by ITS-PCR/RFLP using the restriction enzymes TaqI and MseI. The results obtained indicate that ITS-PCR and ITS-PCR/RFLP assays could be used not only for interspecific, but also for intraspecific, typing.

Differentiation of Cronobacter spp. by tryptic digestion of the cell suspension followed by MALDI-TOF MS analysis

Intact cell MALDI-TOF mass spectrometry is a rapid tool for the identification and classification of microorganisms, now widely used even in clinical laboratories. However, its distinctive power is not sufficient for some closely-related species. The genus Cronobacter, formerly known as Enterobacter sakazakii, contains such species. In this work, a new method for the differentiation of five Cronobacter species is presented involving the tryptic digestion of cytoplasmatic proteins followed by MALDI mass spectrometry analysis. A database was developed for use in Bruker Biotyper software including 52 reference spectra and tested on a set of 45 samples with an overall accuracy of about 80%. The possibility of measurement automation and the short time and low cost requirements of this method compared to those of biochemical tests or PCR methods make it a supplementary option to intact cell MALDI, providing additional information about the differentiation of problematic species.

Characterization of fungal antagonistic bacilli isolated from aerial roots of banyan (Ficus benghalensis) using intact-cell MALDI-TOF mass spectrometry (ICMS)

To characterize fungal antagonistic bacilli isolated from aerial roots of banyan tree and identify the metabolites responsible for their antifungal activity. Seven gram positive, endospore-forming, rod-shaped endophytic bacterial strains exhibiting a broad-spectrum antifungal activity were isolated from the surface-sterilized aerial roots of banyan tree. The isolates designated as K1, A2, A4 and A12 were identified as Bacillus subtilis, whereas isolates A11 and A13 were identified as Bacillus amyloliquefaciens using Biolog Microbial Identification System. The antifungal lipopeptides, surfactins, iturins and fengycins with masses varying in the range from m/z 900 to m/z 1550 could be detected using intact-cell MALDI-TOF mass spectrometry (ICMS). On the basis of mass spectral and carbon source utilization profile, all seven endophytes could be distinguished from each other. Furthermore, ICMS analysis revealed higher extent of heterogeneity among iturins and fengycins produced by B. subtilis K1, correlating well with its higher antifungal activity in comparison with other isolates. Seven fungal antagonistic bacilli were isolated from aerial roots of banyan tree, exhibiting broad spectrum of antifungal activity, among which B. subtilis K1 isolate was found to be most potent. The ICMS analysis revealed that all these isolates produced cyclic lipopeptides belonging to surfactin, iturin and fengycin families and exhibited varying degree of heterogeneity. The endophytes are considered as a potential source of novel bioactive metabolites, and this study describes the potent fungal antagonistic bacilli from aerial roots of banyan tree. The isolates described in this study have a prospective application as biocontrol agents. Also ICMS analysis described in this study for characterization of antifungal metabolites produced by banyan endophytic bacilli may be used as a high throughput tool for screening of microbes producing novel cyclic lipopeptides.

Monitoring virus-like particle and viral protein production by intact cell MALDI-TOF mass spectrometry

A new application of intact cell MALDI- TOF MS ( ICM- MS) methodology is described for monitoring the production of viral proteins and viral like particles using the baculovirus/insect cells expression system. Various MALDI matrices, cell preparation methods, cell/matrix volume ratio and MALDI target application procedures were tested in order to obtain the highest intensity and reproducibility of intact insect cell spectra. The web interface, SPECLUST ( http://bioinfo.thep.lu.se/speclust.html), was used to construct dendograms based on MALDI-TOF MS data for evaluation of fingerprint changes. We demonstrate that insect cell mass spectrum fingerprints are characteristic of each viral protein/particle production. Their changes along the time for each production experiment correlate with the intracellular viral protein content determined by Western blot. This work shows that this simple, fast and low cost assay, which requires low sample volume, is a powerful analytical tool that complements the most common analytical methods used for monitoring bioprocesses and has potential application in the biotechnological industry namely, in the production of recombinant proteins.

Rapid identification of Burkholderia cepacia complex species including strains of the novel Taxon K, recovered from cystic fibrosis patients by intact cell MALDI-ToF mass spectrometry

Two approaches based on intact cell matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (IC-MALDI-ToF MS) have been evaluated in order to discriminate and identify nine former Burkholderia cepacia complex (Bcc) species, Burkholderia contaminans belonging to the novel Taxon K, Burkholderia gladioli, and the most relevant non-fermentative (NF) Gram-negative rods recovered from cystic fibrosis (CF) sputum cultures. In total, 146 clinical isolates and 26 reference strains were analysed. IC mass spectra were obtained with high reproducibility applying a recently developed inactivation protocol which is based on the extraction of microbial proteins by trifluoroacetic acid (TFA). In a first approach, spectral analysis was carried out by means of a gel-view representation of mass spectra, which turned out to be useful to recognize specific identifying biomarker proteins (SIBPs). A series of prominent mass peaks, mainly assigned to constitutively expressed proteins, were selected as SIBPs for identifications at the genus and species level. Two distinctive mass peaks present in B. contaminans spectra (7501 and 7900 Da) were proposed as SIBPs for the identification of this novel species. A second approach of spectral analysis based on data reduction, feature selection and subsequent hierarchical cluster analysis was used to obtain an objective discrimination of all species analysed. Both complementary modalities of analyzing complex IC-MALDI-ToF MS data open the path towards a rapid, accurate and objective means of routine clinical microbiology diagnosis of pathogens from sputum samples of CF patients.

Peptaibiomics: Towards a Myriad of Bioactive Peptides Containing Cα‐Dialkylamino Acids?

Fungi are generally regarded as a literally infinite resource of bioactive secondary metabolites displaying remarkable structural diversity. Research on a particular group of linear and cyclic peptide antibiotics comprehensively named peptaibiotics--as result of the abundance of the non-proteinogenic C(alpha)-dialkylated alpha-amino acids alpha-aminoisobutyric acid (Aib) and isovaline (Iva)--has been started 50 years ago. These peptides have gained constantly increasing interest because of their unique bioactivities and conformations. This review, reflecting the history of peptaibiotic research from 1958 to 2008, is focussed on introducing both the structural diversity and natural microheterogeneity of the peptaibiotics, as well as the biodiversity of their fungal producers. Recently introduced state-of-the-art methods for rapid screening and sequencing of peptaibiotics, such as peptaibiomics and intact-cell MALDI-TOF mass spectrometry, are discussed. Finally, future prospects in peptaibiotic research are presented. Owing to the ubiquity and biodiversity of the fungal producers in the biosphere, the discovery of a myriad of peptaibiotics within the next decade is predicted.

Intact-cell MALDI-TOF mass spectrometry analysis of peptaibol formation by the genus Trichoderma/Hypocrea: can molecular phylogeny of species predict peptaibol structures?

Peptaibols are characteristic linear alpha-aminoisobutyrate-containing peptides produced by certain Ascomycetes, especially of the genus Hypocrea/Trichoderma [Hypocrea and Trichoderma are the names for the teleo- and anamorph forms of the same taxon; where known to occur in nature, the teleomorph is used to name the species. To aid the inexperienced reader, both names (the less well known one in parentheses) are given at the first mention of each species.] Here we have investigated whether phylogenetic relationships within Trichoderma permit a prediction of the peptaibol production profiles. To this end, representative strains from a third (28) of the known species of Trichoderma, identified by the sequences of diagnostic genes and covering most clades of the established multilocus phylogeny of Trichoderma/Hypocrea, were investigated by intact-cell MALDI-TOF mass spectrometry. Peptaibols were detected in all strains, and some strains were found to produce up to five peptide families of different sizes. Comparison of the data with phylogenies derived from rRNA spacer regions (ITS1 and 2) and RNA polymerase subunit B (rpb2) gene sequences did not show a strict correlation with the types and sequences of the peptaibols produced, but the production of some groups of peptaibols appears to be found only in some clades or sections of the genus, which could be used for more targeted screening of novel compounds of this type. In an analysis of peptaibol structures, we have defined conserved key positions and have further identified and compared sequences of the corresponding adenylate domains within non-ribosomal peptide synthetases producing trichovirins, paracelsins and atroviridins. These phylogenies are not concordant with those of their producers Hypocrea virens, Hypocrea jecorina and Hypocrea atroviridis as obtained from ITS1 and 2, and rpb2, respectively, and therefore hint at a complex history of peptaibol diversity.

Rapid screening and dereplication of bacterial isolates from marine sponges of the Sula Ridge by Intact-Cell-MALDI-TOF mass spectrometry (ICM-MS)

Rapid grouping of bacterial isolates is critical in comprehensive microbial studies of environmental samples or screening programmes e.g. in unknown marine environments where large numbers of strains have to be isolated on different growth media. Sets of bacteria have been cultured from the marine sponges Isops phlegraei, Haliclona sp. 1, Phakellia ventilabrum and Plakortis sp. growing at a depth of about 300 m on the Sula Ridge close to the Norwegian coast. We employed Intact-Cell MALDI-TOF (ICM) mass spectrometry to achieve a rapid proteometric clustering of a subset of the strain collection including 456 isolates. Cluster analysis of mass spectra resolved the strains into 11 groups corresponding to species of Alteromonas (15), Bacillus (3), Colwellia (31), Erythrobacter (19), Marinobacter (14), Marinococcus (6), Pseudoalteromonas (297), Pseudomonas (56), Roseobacter (3), Sphingomonas (2) and Vibrio (10) as verified by 16 S rDNA analysis. A further discrimination into subgroups was demonstrated for different isolates from the genus Pseudoalteromonas. The approach described here permits the rapid identification of isolates for dereplication, and the selection of strains representing rare species for subsequent characterization.