Accurate Identification of Closely Related Mycobacterium tuberculosis Complex Species by High Resolution Tandem Mass Spectrometry

Rapid and accurate differentiation of Mycobacterium tuberculosis complex (MTBC) species from other mycobacterium is essential for appropriate therapeutic management, timely intervention for infection control and initiation of appropriate health care measures. However, routine clinical characterization methods for Mycobacterium tuberculosis (Mtb) species remain both, time consuming and labor intensive. In the present study, an innovative liquid Chromatography-Mass Spectrometry method for the identification of clinically most relevant Mycobacterium tuberculosis complex species is tested using a model set of mycobacterium strains. The methodology is based on protein profiling of Mycobacterium tuberculosis complex isolates, which are used as markers of differentiation. To test the resolving power, speed, and accuracy of the method, four ATCC type strains and 37 recent clinical isolates of closely related species were analyzed using this new approach. Using different deconvolution algorithms, we detected hundreds of individual protein masses, with a subpopulation of these functioning as species-specific markers. This assay identified 216, 260, 222, and 201 proteoforms for M. tuberculosis ATCC 27294™, M. microti ATCC 19422™, M. africanum ATCC 25420™, and M. bovis ATCC 19210™ respectively. All clinical strains were identified to the correct species with a mean of 95% accuracy. Our study successfully demonstrates applicability of this novel mass spectrometric approach to identify clinically relevant Mycobacterium tuberculosis complex species that are very closely related and difficult to differentiate with currently existing methods. Here, we present the first proof-of-principle study employing a fast mass spectrometry-based method to identify the clinically most prevalent species within the Mycobacterium tuberculosis species complex.

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ntra-species profiling of Cleome viscosa growing in Swat district (Pakistan)

Biosystems Diversity ◽

10.15421/011808 ◽

2018 ◽

Vol 26 (1) ◽

pp. 52-55

Author(s):

N. Muhammad ◽

S. F. Wadood ◽

W. Khan ◽

N. Ali ◽

M. Nisar

Keyword(s):

Allelic Variation ◽

Lower Surface ◽

Protein Profiling ◽

Yellow Colour ◽

As Species ◽

Sds Page ◽

Specific Variation ◽

Species Specific ◽

High Level ◽

Cleome Viscosa

Intra-specific genetic variation was studied in 28 genotypes of Cleome viscosa L. growing in Swat district, Khyber Pakhtunkhwa, Pakistan. It was found that genotypes showed the utmost allelic variation for leaf upper and lower surface with emerald green (75%), and yellow green (75%) respectively, other leaves lower and upper surfaces were (25%) green and yellow green (26%) respectively. The majority of C. viscosa genotypes were (50%) yellow flowers while others were with (29%) white yellow colour and (21%) dull yellow. Most of the seeds were with black (46%). The protein profiling was carried out on 12% gel electrophoresis; seven reproducible bands with molecular weight ranges from 180 to 10 KDa were detected in C. viscosa, the locus contribution toward genetic disagreement (LCTGD) of C. viscosa was 57%. Notably, L-3, L-4 L-5, was monomorphic in C. viscosa and was treated as species specific. L-1, L-2, L-7 were polymorphic. These bands showed 79%, 4%, 14% and 79% variation respectively. In the current investigation the intra-specific variation was observed limited and alone SDS-PAGE did not determine the high level of intra-specific variation; however, diverse germplasm were suggested to be acquired from various sources.

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Genetic diversity in the natural population of threatened fruit- bearing plant species Monotheca buxifolia (Falc.) A.D.

Ukrainian Journal of Ecology ◽

10.15421/2020_204 ◽

2020 ◽

Vol 10 (5) ◽

pp. 44-48

Author(s):

M. Noor ◽

U. Nisar ◽

K.U.K. Muhhamad

Keyword(s):

Genetic Diversity ◽

Plant Species ◽

Gel Electrophoresis ◽

Sodium Dodecyl ◽

Plant Conservation ◽

Protein Profiling ◽

As Species ◽

Sds Page ◽

Monotheca Buxifolia ◽

Species Specific

Monotheca buxifolia is an ethnomedicinally and economically important threatened fruit bearing plant species in Malakand Division Pakistan. The genetic diversity among the 92 various genotypes of Monotheca buxifolia was carried out using sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE) method. A considerable amount of inter districts genetic diversity (66.70%) was observed among the genotypes of M. buxifolia. Protein profiling was conducted on 12% gel electrophoresis. A total of 6 protein bands were observed in M. buxifolia genotypes. SDS-PAGE practice is a convenient scheme for the examination of both genetic diversity and relationship. Particularly, L-4 and L-5 were monomorphic in the inter districts Monotheca buxifolia genotypes and was recognized as species specific. The remaining other loci were polymorphic. In this investigation, the high inter and intra- districts specific diversity was observed demonstrating SDS-PAGE is an authoritative procedure for categorizing the genetically diverse germplasms in M. buxifolia. The findings from this study could be useful in the identification and selection of suitable M. buxifolia genotypes for future conservation programmes. Today, there is still a need to examine the genetic diversity and protect genetic resources, in particular wild species, for possible benefits in plant conservation programmes. To the best of our knowledge, this is the first ever report that addresses genetic variability in M. buxifolia.

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A New Accurate Genotyping HRM Method for Alternaria Species Related to Fruit Rot Diseases of Apple and Pomegranate

International Journal of Phytopathology ◽

10.33687/phytopath.004.03.1531 ◽

2016 ◽

Vol 4 (3) ◽

pp. 159-165 ◽

Cited By ~ 1

Author(s):

Antonios Zambounis ◽

Aliki Xanthopoulou ◽

George Karaoglanidis ◽

Athanasios Tsaftaris ◽

Panagiotis Madesis

Keyword(s):

Morphological Characteristics ◽

Curve Analysis ◽

Fruit Rot ◽

Nucleotide Polymorphisms ◽

Postharvest Diseases ◽

Accurate Identification ◽

Complex Species ◽

Alternaria Species ◽

Species Specific ◽

Pomegranate Fruit

Alternaria core rot and Alternaria black heart rot of apple and pomegranate fruit, respectively, are major pre- and postharvest diseases worldwide. However, it is very difficult to differentiate the rot related Alternaria species in the Alternaria complex as they are not always correlate to species-groups based upon morphological characteristics and due to the limited genetic variation these species exhibit among each other. Therefore, it is crucial to exploit novel assays towards the accurate identification and differentiation of these Alternaria species. We have developed, a real-time PCR assay [using species specific primers targeting the endopolygalacturonase (EndoPG) gene] combined with a high-resolution melting (HRM) curve analysis for discrimination of the 14 single nucleotide polymorphisms (SNPs)-based Alternaria haplotypes, which were assigned based on the aligned sequence profiles of 138 Alternaria spp. strains previously isolated from apple and pomegranate rotted fruit. This analysis specifically generated 14 unique HRM curve haplotype profiles among the Alternaria complex species tested. The results showed that HRM curve analysis allows the rapid and adequate identification and genotyping of the three Alternaria species (A. alternata, A. tenuissima and A. arborescens) responsible mostly for the apple and pomegranate fruit rot diseases.

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Protein profiling and analysis of drug sensitive and multidrug resistant isolates of Mycobacterium tuberculosis by native polyacrylamide gel electrophoresis and mass spectrometry

Vaccine Research ◽

10.18869/acadpub.vacres.2.4.81 ◽

2015 ◽

Vol 2 (4) ◽

pp. 81-85

Author(s):

Sh Yari ◽

AR Hadizadeh Tasbiti ◽

M Ghanei ◽

MA Shokrgozar ◽

R Mahdian ◽

...

Keyword(s):

Mass Spectrometry ◽

Mycobacterium Tuberculosis ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Multidrug Resistant ◽

Polyacrylamide Gel ◽

Protein Profiling ◽

Native Polyacrylamide Gel Electrophoresis

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Ultra-High-Resolution Mass Spectrometry for Identification of Closely Related Dermatophytes with Different Clinical Predilections

Journal of Clinical Microbiology ◽

10.1128/jcm.00102-18 ◽

2018 ◽

Vol 56 (7) ◽

Cited By ~ 7

Author(s):

Karolina Dukik ◽

Joanna Freeke ◽

Azadeh Jamalian ◽

Bert Gerrits van den Ende ◽

Ping Yip ◽

...

Keyword(s):

Mass Spectrometry ◽

High Resolution Mass Spectrometry ◽

Single Species ◽

Strain Level ◽

Resolving Power ◽

Microbial Pathogens ◽

Content Type ◽

Flight Mass Spectrometry ◽

Liquid Chromatography Tandem Mass ◽

Species Specific

ABSTRACT In the present study, an innovative top-down liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the identification of clinically relevant fungi is tested using a model set of dermatophyte strains. The methodology characterizes intact proteins derived from Trichophyton species, which are used as parameters of differentiation. To test its resolving power compared to that of traditional Sanger sequencing and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF), 24 strains of closely related dermatophytes, Trichophyton rubrum , T. violaceum , T. tonsurans , T. equinum , and T. interdigitale , were subjected to this new approach. Using MS/MS and different deconvolution algorithms, we identified hundreds of individual proteins, with a subpopulation of these used as strain- or species-specific markers. Three species, i.e., T. rubrum , T. violaceum , and T. interdigitale , were identified correctly down to the species level. Moreover, all isolates associated with these three species were identified correctly down to the strain level. In the T. tonsurans - equinum complex, eight out of 12 strains showed nearly identical proteomes, indicating an unresolved taxonomic conflict already apparent from previous phylogenetic data. In this case, it was determined with high probability that only a single species can be present. Our study successfully demonstrates applicability of the mass spectrometric approach to identify clinically relevant filamentous fungi. Here, we present the first proof-of-principle study employing the mentioned technology to differentiate microbial pathogens. The ability to differentiate fungi at the strain level sets the stage to improve patient outcomes, such as early detection of strains that carry resistance to antifungals.

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Speciation of the Mycobacterium tuberculosis complex using mass spectrometry: a proof-of-concept

10.1101/2021.02.04.429756 ◽

2021 ◽

Author(s):

Simon Robinne ◽

Jamal Saad ◽

Madjid Morsli ◽

Hamidou Zelika Harouna ◽

Fatah Tazerart ◽

...

Keyword(s):

Mass Spectrometry ◽

Mycobacterium Tuberculosis ◽

Dna Analysis ◽

Species Level ◽

Complex Species ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Source Tracing ◽

Tof Ms

ABSTRACTMycobacteria that form the Mycobacterium tuberculosis complex are responsible for deadly tuberculosis in animals and patients. Identification of these pathogens at the species level is of primary importance for treatment and source tracing, and currently relies on DNA analysis, including whole genome sequencing (WGS), which takes a whole day. In this study, we report on the unprecedented identification of the M. tuberculosis complex species using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), with WGS as the comparative gold standard. In a first step, an optimised peptide extraction applied to 24 isolates otherwise identified in three of the 11 M. tuberculosis complex species by WGS, yielded 94 MALDI-TOF spectra which clustered according to WGS identification. In a second step, 70/74 (95%) other isolates were correctly identified at the species level by this clustering method. This study is the first to report a MALDI-TOF-MS method of identification of M. tuberculosis complex mycobacteria at the species level and is easily implantable in clinical microbiology laboratories.

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Spatial Metabolomics of the Human Kidney Using MALDI Trapped Ion Mobility Imaging Mass Spectrometry

10.26434/chemrxiv.12118644.v2 ◽

2020 ◽

Author(s):

Elizabeth Neumann ◽

Lukasz Migas ◽

Jamie L. Allen ◽

Richard Caprioli ◽

Raf Van de Plas ◽

...

Keyword(s):

Mass Spectrometry ◽

Ion Mobility ◽

Imaging Mass Spectrometry ◽

Structural Complexity ◽

Human Kidney ◽

Resolving Power ◽

Mobility Separation ◽

Trapped Ion ◽

Mobility Data ◽

Ion Mobility Separation

<div> <div> <p>Small metabolites are essential for normal and diseased biological function but are difficult to study because of their inherent structural complexity. MALDI imaging mass spectrometry (IMS) of small metabolites is particularly challenging as MALDI matrix clusters are often isobaric with metabolite ions, requiring high resolving power instrumentation or derivatization to circumvent this issue. An alternative to this is to perform ion mobility separation before ion detection, enabling the visualization of metabolites without the interference of matrix ions. Here, we use MALDI timsTOF IMS to image small metabolites at high spatial resolution within the human kidney. Through this, we have found metabolites, such as arginic acid, acetylcarnitine, and choline that localize to the cortex, medulla, and renal pelvis, respectively. We have also demonstrated that trapped ion mobility spectrometry (TIMS) can resolve matrix peaks from metabolite signal and separate both isobaric and isomeric metabolites with different localizations within the kidney. The added ion mobility data dimension dramatically increased the peak capacity for molecular imaging experiments. Future work will involve further exploring the small metabolite profiles of human kidneys as a function of age, gender, and ethnicity.</p></div></div>

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Suzuki–Miyaura cross-coupling for chemoproteomic applications

10.26434/chemrxiv.12055218.v1 ◽

2020 ◽

Author(s):

Jian Cao ◽

Ernest Armenta ◽

Lisa Boatner ◽

Heta Desai ◽

Neil Chan ◽

...

Keyword(s):

Mass Spectrometry ◽

Cross Coupling ◽

Protein Profiling ◽

Protein Labeling ◽

Caspase 8 ◽

Complex Cell ◽

Bioorthogonal Chemistry ◽

Reaction Conditions ◽

Target Deconvolution ◽

Palladium Catalyzed

Bioorthogonal chemistry is a mainstay of chemoproteomic sample preparation workflows. While numerous transformations are now available, chemoproteomic studies still rely overwhelmingly on copper-catalyzed azide –alkyne cycloaddition (CuAAC) or 'click' chemistry. Here we demonstrate that gel-based activity-based protein profiling (ABPP) and mass-spectrometry-based chemoproteomic profiling can be conducted using Suzuki–Miyaura cross-coupling. We identify reaction conditions that proceed in complex cell lysates and find that Suzuki –Miyaura cross-coupling and CuAAC yield comparable chemoproteomic coverage. Importantly, Suzuki–Miyaura is also compatible with chemoproteomic target deconvolution, as demonstrated using structurally matched probes tailored to react with the cysteine protease caspase-8. Uniquely enabled by the observed orthogonality of palladium-catalyzed cross-coupling and CuAAC, we combine both reactions to achieve dual protein labeling.

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Arsenic speciation in rice and risk assessment of inorganic arsenic from Ghentugachhi village of Chakdaha block, Nadia, West Bengal, India

ORYZA- An International Journal on Rice ◽

10.35709/ory.2020.57.2.1 ◽

2020 ◽

Vol 57 (2) ◽

pp. 85-93

Author(s):

B Sinha ◽

K Bhattacharyya

Keyword(s):

Mass Spectrometry ◽

Inductively Coupled Plasma ◽

West Bengal ◽

Arsenic Speciation ◽

Inorganic Arsenic ◽

Rice Grain ◽

Species Determination ◽

Inductively Coupled ◽

As Species ◽

Plasma Mass Spectrometry

The purpose of the present study was to assess arsenic (As) speciation in rice from West Bengal, India, in order to improve understanding of the health risk posed by arsenic in Indian rice. Rice is a potentially important route of human exposure to arsenic, especially in populations with rice-based diets. However, arsenic toxicity varies greatly with species. Determination of arsenic (As) species in rice is necessary because inorganic As species are more toxic than organic As. Total arsenic was determined by inductively coupled plasma mass spectrometry; arsenite, arsenate, monomethylarsonic acid, and dimethyarsinic acid were quantified by high-performance liquid chromatography- inductively coupled plasma mass spectrometry. The analysis of a rice flour certified reference material (SRM-1568-a) were evaluated for quality assurance. The use of 2M TFA for extraction with an isocratic mobile phase was optimized for extraction and employed for arsenic speciation in rice. The extraction method showed a high recovery of arsenic. Most of the As species in rice were noticed to be inorganic [Arsenite (As-III), Arsenate As-V]. It appeared very clear from the present study that inorganic arsenic shared maximum arsenic load in rice straw while in grains it is considerably low. As species recovered from rice grain and straw are principally As-III and As-V with a little share of DMA and almost non-detectable MMA and As-B. The order of As species in rice grain revealed in this study were As-III (54.5-65.4 %)>As-V(21.2-28.3%)>DMA(5.2%).

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