15 research outputs found
Further development of high-sensitivity targeted MRM Assays for peptide and protein quantification
Comunicaciones a congreso
Profiling invasive Plasmodium falciparum merozoites using an integrated omics approach
The symptoms of malaria are brought about by blood-stage parasites, which are established when merozoites invade human erythrocytes. Our understanding of the molecular events that underpin erythrocyte invasion remains hampered by the short-period of time that merozoites are invasive. To address this challenge, a Plasmodium falciparum gamma-irradiated long-lived merozoite (LLM) line was developed and investigated. Purified LLMs invaded erythrocytes by an increase of 10–300 fold compared to wild-type (WT) merozoites. Using an integrated omics approach, we investigated the basis for the phenotypic difference. Only a few single nucleotide polymorphisms within the P. falciparum genome were identified and only marginal differences were observed in the merozoite transcriptomes. By contrast, using label-free quantitative mass-spectrometry, a significant change in protein abundance was noted, of which 200 were proteins of unknown function. We determined the relative molar abundance of over 1100 proteins in LLMs and further characterized the major merozoite surface protein complex. A unique processed MSP1 intermediate was identified in LLM but not observed in WT suggesting that delayed processing may be important for the observed phenotype. This integrated approach has demonstrated the significant role of the merozoite proteome during erythrocyte invasion, while identifying numerous unknown proteins likely to be involved in invasion
Design and application of a data-independent precursor and product ion repository
The functional design and application of a data-independent LC-MS precursor and product ion repository for protein identification, quantification, and validation is conceptually described. The ion repository was constructed from the sequence search results of a broad range of discovery experiments investigating various tissue types of two closely related mammalian species. The relative high degree of similarity in protein complement, ion detection, and peptide and protein identification allows for the analysis of normalized precursor and product ion intensity values, as well as standardized retention times, creating a multidimensional/orthogonal queryable, qualitative, and quantitative space. Peptide ion map selection for identification and quantification is primarily based on replication and limited variation. The information is stored in a relational database and is used to create peptide- and protein-specific fragment ion maps that can be queried in a targeted fashion against the raw or time aligned ion detections. These queries can be conducted either individually or as groups, where the latter affords pathway and molecular machinery analysis of the protein complement. The presented results also suggest that peptide ionization and fragmentation efficiencies are highly conserved between experiments and practically independent of the analyzed biological sample when using similar instrumentation. Moreover, the data illustrate only minor variation in ionization efficiency with amino acid sequence substitutions occurring between species. Finally, the data and the presented results illustrate how LC-MS performance metrics can be extracted and utilized to ensure optimal performance of the employed analytical workflows
Scanning Quadrupole Data-Independent Acquisition, Part B: Application to the Analysis of the Calcineurin-Interacting Proteins during Treatment of <i>Aspergillus fumigatus</i> with Azole and Echinocandin Antifungal Drugs
Calcineurin is a critical cell-signaling
protein that orchestrates
growth, stress response, virulence, and antifungal drug resistance
in several fungal pathogens. Blocking calcineurin signaling increases
the efficacy of several currently available antifungals and suppresses
drug resistance. We demonstrate the application of a novel scanning
quadrupole DIA method for the analysis of changes in the proteins
coimmunoprecipitated with calcineurin during therapeutic antifungal
drug treatments of the deadly human fungal pathogen <i>Aspergillus
fumigatus.</i> Our experimental design afforded an assessment
of the precision of the method as demonstrated by peptide- and protein-centric
analysis from eight replicates of the study pool QC samples. Two distinct
classes of clinically relevant antifungal drugs that are guideline
recommended for the treatment of invasive “aspergillosis”
caused by <i>Aspergillus fumigatus</i>, the azoles (voriconazole)
and the echinocandins (caspofungin and micafungin), which specifically
target the fungal plasma membrane and the fungal cell wall, respectively,
were chosen to distinguish variations occurring in the proteins coimmunoprecipitated
with calcineurin. Novel potential interactors were identified in response
to the different drug treatments that are indicative of the possible
role for calcineurin in regulating these effectors. Notably, treatment
with voriconazole showed increased immunoprecipitation of key proteins
involved in membrane ergosterol biosynthesis with calcineurin. In
contrast, echinocandin (caspofungin or micafungin) treatments caused
increased immunoprecipitation of proteins involved in cell-wall biosynthesis
and septation. Furthermore, abundant coimmunoprecipitation of ribosomal
proteins with calcineurin occurred exclusively in echinocandins treatment,
indicating reprogramming of cellular growth mechanisms during different
antifungal drug treatments. While variations in the observed calcineurin
immunoprecipitated proteins may also be due to changes in their expression
levels under different drug treatments, this study suggests an important
role for calcineurin-dependent cellular mechanisms in response to
antifungal treatment of <i>A.</i> fumigatus that warrants
future studies
Scanning Quadrupole Data-Independent Acquisition, Part A: Qualitative and Quantitative Characterization
A novel data-independent
acquisition (DIA) method incorporating
a scanning quadrupole in front of a collision cell and orthogonal
acceleration time-of-flight mass analyzer is described. The method
has been characterized for the qualitative and quantitative label-free
proteomic analysis of complex biological samples. The principle of
the scanning quadrupole DIA method is discussed, and analytical instrument
characteristics, such as the quadrupole transmission width, scan/integration
time, and chromatographic separation, have been optimized in relation
to sample complexity for a number of different model proteomes of
varying complexity and dynamic range including human plasma, cell
lines, and bacteria. In addition, the technological merits over existing
DIA approaches are described and contrasted. The qualitative and semiquantitative
performance of the method is illustrated for the analysis of relatively
simple protein digest mixtures and a well-characterized human cell
line sample using untargeted and targeted search strategies. Finally,
the results from a human cell line were compared against publicly
available data that used similar chromatographic conditions but were
acquired with DDA technology and alternative mass analyzer systems.
Qualitative comparison showed excellent concordance of results with
>90% overlap of the detected proteins
Unfold thy snowy pinions [music] : a Maori love song /
119784 (Publisher number). Caption title.; For voice and piano.; Pl. no.: 119784.; "No. 1 in F min"--Cover.; Also available online http://nla.gov.au/nla.mus-vn3889726