An Efficient In-gel Digestion Protocol for Mass Spectral Analysis by MALDI-TOF-MS and MS/MS and Its Use for Proteomic Analysis of Vigna mungo Leaves

Background: Most of the proteomic studies in Escherichia coli have focussed on pathogenic strains, while very few studies have studied the commensal strains. It is important to study the commensal strains because under the selective pressure of their habitat, commensal strains might serve as reservoirs of virulent and pathogenic strains. Objective: In this study, we have performed a comparative proteomic analysis of commensal and pathogenic strains of E. coli isolated from a major river flowing through northern India. Methods: Proteins were resolved by two dimensional gel electrophoresis and the differentially expressed proteins were identified using matrix-assisted laser desorption ionization-time of flight mass-spectrometry (MALDI-TOF MS). Results: Many proteins of the commensal strain showed an increased expression compared to the pathogenic strain, of which seventeen proteins were identified by MALDI-TOF MS. Functional classification of these proteins revealed that they belonged to different functional pathways like energy metabolism, nucleotide and nucleoside conversions, translation, biosynthesis of amino acids and motility and energytaxis/chemotaxis. Conclusion: As per the best of our knowledge, this is the first report on comparative proteomic analysis of E. coli commensal and pathogenic strains of aquatic origin. Our results suggest that the increased production of these proteins might play an important role in adaptation of E. coli to a commensal/pathogenic lifestyle. However, further experiments are required to understand the precise role of these proteins in regulating the pathogenicity/commensalism of E. coli.

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Proteomic analysis of injured spinal cord tissue proteins using 2-DE and MALDI-TOF MS

PROTEOMICS ◽

10.1002/pmic.200500621 ◽

2006 ◽

Vol 6 (9) ◽

pp. 2797-2812 ◽

Cited By ~ 46

Author(s):

Soo Kyung Kang ◽

Hyeun Hwa So ◽

Yo Seup Moon ◽

Cheul Hong Kim

Keyword(s):

Spinal Cord ◽

Proteomic Analysis ◽

Spinal Cord Tissue ◽

Injured Spinal Cord ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

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Prognostic accuracy of MALDI mass spectrometric analysis of plasma in COVID-19

10.1101/2020.10.01.20205310 ◽

2020 ◽

Cited By ~ 1

Author(s):

Lucas Cardoso Lazari ◽

Fabio De Rose Ghilardi ◽

Livia Rosa-Fernandes ◽

Diego M Assis ◽

José Carlos Nicolau ◽

...

Keyword(s):

Plasma Protein ◽

Proteomic Analysis ◽

Serum Amyloid A ◽

Low Risk ◽

Serum Amyloid ◽

Bottom Up ◽

Maldi Tof Ms ◽

Amyloid A ◽

Maldi Tof ◽

Tof Ms

AbstractPurposeSARS-CoV-2 infection poses a global public health problem. There is a critical need for improvements in the noninvasive prognosis of COVID-19. We hypothesized that matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS) analysis combined with bottom-up proteomic analysis of plasma proteins might identify features to predict high and low risk cases of COVID-19.Patients and MethodsWe used MALDI-TOF MS to analyze plasma small proteins and peptides isolated using C18 micro-columns from a cohort containing a total of 117 cases of high (hospitalized) and low risk (outpatients) cases split into training (n = 88) and validation sets (n= 29). The plasma protein/peptide fingerprint obtained was used to train the algorithm before validation using a blinded test cohort.ResultsSeveral sample preparation, MS and data analysis parameters were optimized to achieve an overall accuracy of 85%, sensitivity of 90%, and specificity of 81% in the training set. In the blinded test set, this signature reached an overall accuracy of 93.1%, sensitivity of 87.5%, and specificity of 100%. From this signature, we identified two distinct regions in the MALDI-TOF profile belonging to the same proteoforms. A combination of 1D SDS-PAGE and quantitative bottom-up proteomic analysis allowed the identification of intact and truncated forms of serum amyloid A-1 and A-2 proteins. Conclusions: We found a plasma proteomic profile that discriminates against patients with high and low risk COVID-19. Proteomic analysis of C18-fractionated plasma may have a role in the noninvasive prognosis of COVID-19. Further validation will consolidate its clinical utility.Key messageWhat is the key question?Do individuals infected with SARS-CoV-2 harboring different degree of disease severity have a plasma protein profile that differentiate them and predict the COVID-19 outcome?What is the bottom line?In a series of 117 patients with COVID-19 divided in hospitalized (60) and outpatients (57), differential expression of serum amyloid A-1 (SAA1) and A-2 (SAA2) predict their outcome.Why read on?The high mortality rate in SARS-CoV-2 infected individuals requires accurate markers for predicting COVID-19 severity. Plasma levels of SAA1 and SAA2 indicate higher risk of hospitalization and can be used to improve COVID-19 monitoring and therapy.

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Factors Associated With MALDI-TOF Mass Spectral Quality of Species Identification in Clinical Routine Diagnostics

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.646648 ◽

2021 ◽

Vol 11 ◽

Author(s):

Aline Cuénod ◽

Frédéric Foucault ◽

Valentin Pflüger ◽

Adrian Egli

Keyword(s):

Species Identification ◽

Burkholderia Cepacia ◽

Bacterial Species ◽

Clinical Diagnostics ◽

Spectral Quality ◽

Bacterial Colony ◽

Maldi Tof Ms ◽

Mass Spectral ◽

Maldi Tof ◽

Tof Ms

BackgroundAn accurate and timely identification of bacterial species is critical in clinical diagnostics. Species identification allows a potential first adaptation of empiric antibiotic treatments before the resistance profile is available. Matrix assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-TOF MS) is a widely used method for bacterial species identification. However, important challenges in species identification remain. These arise from (i) incomplete databases, (ii) close relatedness of species of interest, and (iii) spectral quality, which is currently vaguely defined.MethodsWe selected 47 clinically relevant bacterial isolates from 39 species, which can be challenging to identify by MALDI-TOF MS. We measured these isolates under various analytical conditions on two MALDI-TOF MS systems. First, we identified spectral features, which were associated with correct species identification in three different databases. Considering these features, we then systematically compared spectra produced with three different sample preparation protocols. In addition, we varied quantities of bacterial colony material applied and bacterial colony age.ResultsWe identified (i) the number of ribosomal marker peaks detected, (ii) the median relative intensity of ribosomal marker peaks, (iii) the sum of the intensity of all detected peaks, (iv) a high measurement precision, and (v) reproducibility of peaks to act as good proxies of spectral quality. We found that using formic acid, measuring bacterial colonies at a young age, and frequently calibrating the MALDI-TOF MS device increase mass spectral quality. We further observed significant differences in spectral quality between different bacterial taxa and optimal measurement conditions vary per taxon.ConclusionWe identified and applied quality measures for MALDI-TOF MS and optimized spectral quality in routine settings. Phylogenetic marker peaks can be reproducibly detected and provide an increased resolution and the ability to distinguish between challenging species such as those within the Enterobacter cloacae complex, Burkholderia cepacia complex, or viridans streptococci.

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Proteomic analysis of proteins differentially expressed in conidia and mycelium of the entomopathogenic fungus Aschersonia placenta

Canadian Journal of Microbiology ◽

10.1139/w2012-111 ◽

2012 ◽

Vol 58 (12) ◽

pp. 1327-1334 ◽

Cited By ~ 4

Author(s):

Junzhi Qiu ◽

Yubin Su ◽

Ivan Gelbǐc ◽

Yunfeng Qiu ◽

Xiaocong Xie ◽

...

Keyword(s):

Proteomic Analysis ◽

Entomopathogenic Fungus ◽

Developmental Stages ◽

Differentially Expressed ◽

Uridine Diphosphate ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Oxidation Reduction ◽

Tof Ms

The infection of insects by the entomopathogenic fungus Aschersonia placenta depends on conidia. To identify proteins differentially expressed in A. placenta conidia vs mycelia, we performed a comparative proteomic analysis of A. placenta using 2-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS). We detected 1022 2-DE protein spots in conidia and 1049 in mycelia and analyzed 48 (13 from conidia and 35 from mycelia) using MALDI-TOF-MS. Finally, we identified 28 proteins (7 from conidia and 21 from mycelia). The identified proteins exclusive to conidia included major proteins participating in oxidation–reduction processes and vegetative insecticidal protein 1 (Vip1), a protein that is likely involved in pathogenicity. The identified proteins exclusive to mycelia were those involved in biosynthesis and metabolism, including uridine diphosphate galactopyranose mutase, which might play key roles in hyphal morphogenesis. This report provides the first proteomic analysis of different developmental stages of an Aschersonia species. Although only a small number of proteins were identified, the data represent a useful foundation for future studies concerning the molecular basis of entomopathogenicity in the species A. placenta and in the genus Aschersonia.

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Evaluation of Vitek MS for Differentiation of Cryptococcus neoformans and Cryptococcus gattii Genotypes

Journal of Clinical Microbiology ◽

10.1128/jcm.01282-18 ◽

2018 ◽

Vol 57 (1) ◽

Cited By ~ 2

Author(s):

Lumena P. Machado Siqueira ◽

Viviane M. Favero Gimenes ◽

Roseli Santos de Freitas ◽

Márcia de Souza Carvalho Melhem ◽

Lucas Xavier Bonfietti ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Cryptococcus Gattii ◽

Correct Identification ◽

Content Type ◽

Maldi Tof Ms ◽

Mass Spectral ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Vitek Ms ◽

Tof Ms

ABSTRACT Cryptococcus neoformans and Cryptococcus gattii are the main pathogenic species of invasive cryptococcosis among the Cryptococcus species. Taxonomic studies have shown that these two taxa have different genotypes or molecular types with biological and ecoepidemiological peculiarities. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has been proposed as an alternative method for labor-intensive methods for C. neoformans and C. gattii genotype differentiation. However, Vitek MS, one of the commercial MALDI-TOF MS instruments, has not been yet been evaluated for this purpose. Thus, we constructed an in-house database with reference strains belonging to the different C. neoformans (VNI, VNII, VNIII, and VNIV) and C. gattii (VGI, VGII, VGIII, and VGIV) major molecular types by using the software Saramis Premium (bioMérieux, Marcy-l’Etoile, France). Then, this new database was evaluated for discrimination of the different genotypes. Our in-house database provided correct identification for all C. neoformans and C. gattii genotypes; however, due to the intergenotypic mass spectral similarities, a careful postanalytic evaluation is necessary to provide correct genotype identification.

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