165 research outputs found
Additional file 1 of Multi-Omics integration can be used to rescue metabolic information for some of the dark region of the Pseudomonas putida proteome
Additional file 1
Optimized Extraction Method To Remove Humic Acid Interferences from Soil Samples Prior to Microbial Proteome Measurements
The
microbial composition and their activities in soil environments
play a critical role in organic matter transformation and nutrient
cycling. Liquid chromatography coupled to high-performance mass spectrometry
provides a powerful approach to characterize soil microbiomes; however,
the limited microbial biomass and the presence of abundant interferences
in soil samples present major challenges to proteome extraction and
subsequent MS measurement. To this end, we have designed an experimental
method to improve microbial proteome measurement by removing the soil-borne
humic substances coextraction from soils. Our approach employs an <i>in situ</i> detergent-based microbial lysis/TCA precipitation
coupled to an additional cleanup step involving acidified precipitation
and filtering at the peptide level to remove most of the humic acid
interferences prior to proteolytic peptide measurement. The novelty
of this approach is an integration to exploit two different characteristics
of humic acids: (1) Humic acids are insoluble in acidic solution but
should not be removed at the protein level, as undesirable protein
removal may also occur. Rather it is better to leave the humics acids
in the samples until the peptide level, at which point the significant
differential solubility of humic acids versus peptides at low pH can
be exploited very efficiently. (2) Most of the humic acids have larger
molecule weights than the peptides. Therefore, filtering a pH 2 to
3 peptide solution with a 10 kDa filter will remove most of the humic
acids. This method is easily interfaced with normal proteolytic processing
approaches and provides a reliable and straightforward protein extraction
method that efficiently removes soil-borne humic substances without
inducing proteome sample loss or biasing protein identification in
mass spectrometry. In general, this humic acid removal step is universal
and can be adopted by any workflow to effectively remove humic acids
to avoid them negatively competing with peptides for binding with
reversed-phase resin or ionization in the electrospray
Table_1_Antibiotic resistance and host immune system-induced metal bactericidal control are key factors for microbial persistence in the developing human preterm infant gut microbiome.XLSX
The human gut microbiome, which develops and stabilizes during the early stages of infant life, plays an essential role in host health through the production of metabolic resources and the stimulation and training of the immune system. To study colonization and community functional dynamics of the microbiota based on responses to host immune processes during the normal and dysbiotic establishment of the gut, metaproteomics was conducted on 91 fecal samples collected over the first 90 days of life from 17 hospitalized premature infants. Microbial responses to antibiotic administration and host-imposed metal bactericidal control correlated with community assembly and resiliency of microbes in the developing preterm gut. Specifically, proteins related to antibiotic resistance and metal homeostasis mechanisms were predominant in persisting members in the infant gut environment over the first several weeks of life. Overall, this metaproteomics study provides a unique approach to examine the temporal expansion and resilience of microbial colonization, as it allows simultaneous examination of both host and microbial metabolic activities. Understanding the interplay between host and microbes may elucidate the microbiome’s potential immunomodulatory roles relevant to necrotizing enterocolitis and other dysbiotic conditions in preterm infants.</p
Data_Sheet_1_Antibiotic resistance and host immune system-induced metal bactericidal control are key factors for microbial persistence in the developing human preterm infant gut microbiome.PDF
The human gut microbiome, which develops and stabilizes during the early stages of infant life, plays an essential role in host health through the production of metabolic resources and the stimulation and training of the immune system. To study colonization and community functional dynamics of the microbiota based on responses to host immune processes during the normal and dysbiotic establishment of the gut, metaproteomics was conducted on 91 fecal samples collected over the first 90 days of life from 17 hospitalized premature infants. Microbial responses to antibiotic administration and host-imposed metal bactericidal control correlated with community assembly and resiliency of microbes in the developing preterm gut. Specifically, proteins related to antibiotic resistance and metal homeostasis mechanisms were predominant in persisting members in the infant gut environment over the first several weeks of life. Overall, this metaproteomics study provides a unique approach to examine the temporal expansion and resilience of microbial colonization, as it allows simultaneous examination of both host and microbial metabolic activities. Understanding the interplay between host and microbes may elucidate the microbiome’s potential immunomodulatory roles relevant to necrotizing enterocolitis and other dysbiotic conditions in preterm infants.</p
Table_2_Antibiotic resistance and host immune system-induced metal bactericidal control are key factors for microbial persistence in the developing human preterm infant gut microbiome.XLSX
The human gut microbiome, which develops and stabilizes during the early stages of infant life, plays an essential role in host health through the production of metabolic resources and the stimulation and training of the immune system. To study colonization and community functional dynamics of the microbiota based on responses to host immune processes during the normal and dysbiotic establishment of the gut, metaproteomics was conducted on 91 fecal samples collected over the first 90 days of life from 17 hospitalized premature infants. Microbial responses to antibiotic administration and host-imposed metal bactericidal control correlated with community assembly and resiliency of microbes in the developing preterm gut. Specifically, proteins related to antibiotic resistance and metal homeostasis mechanisms were predominant in persisting members in the infant gut environment over the first several weeks of life. Overall, this metaproteomics study provides a unique approach to examine the temporal expansion and resilience of microbial colonization, as it allows simultaneous examination of both host and microbial metabolic activities. Understanding the interplay between host and microbes may elucidate the microbiome’s potential immunomodulatory roles relevant to necrotizing enterocolitis and other dysbiotic conditions in preterm infants.</p
Table_3_Antibiotic resistance and host immune system-induced metal bactericidal control are key factors for microbial persistence in the developing human preterm infant gut microbiome.XLSX
The human gut microbiome, which develops and stabilizes during the early stages of infant life, plays an essential role in host health through the production of metabolic resources and the stimulation and training of the immune system. To study colonization and community functional dynamics of the microbiota based on responses to host immune processes during the normal and dysbiotic establishment of the gut, metaproteomics was conducted on 91 fecal samples collected over the first 90 days of life from 17 hospitalized premature infants. Microbial responses to antibiotic administration and host-imposed metal bactericidal control correlated with community assembly and resiliency of microbes in the developing preterm gut. Specifically, proteins related to antibiotic resistance and metal homeostasis mechanisms were predominant in persisting members in the infant gut environment over the first several weeks of life. Overall, this metaproteomics study provides a unique approach to examine the temporal expansion and resilience of microbial colonization, as it allows simultaneous examination of both host and microbial metabolic activities. Understanding the interplay between host and microbes may elucidate the microbiome’s potential immunomodulatory roles relevant to necrotizing enterocolitis and other dysbiotic conditions in preterm infants.</p
Table_4_Antibiotic resistance and host immune system-induced metal bactericidal control are key factors for microbial persistence in the developing human preterm infant gut microbiome.XLSX
The human gut microbiome, which develops and stabilizes during the early stages of infant life, plays an essential role in host health through the production of metabolic resources and the stimulation and training of the immune system. To study colonization and community functional dynamics of the microbiota based on responses to host immune processes during the normal and dysbiotic establishment of the gut, metaproteomics was conducted on 91 fecal samples collected over the first 90 days of life from 17 hospitalized premature infants. Microbial responses to antibiotic administration and host-imposed metal bactericidal control correlated with community assembly and resiliency of microbes in the developing preterm gut. Specifically, proteins related to antibiotic resistance and metal homeostasis mechanisms were predominant in persisting members in the infant gut environment over the first several weeks of life. Overall, this metaproteomics study provides a unique approach to examine the temporal expansion and resilience of microbial colonization, as it allows simultaneous examination of both host and microbial metabolic activities. Understanding the interplay between host and microbes may elucidate the microbiome’s potential immunomodulatory roles relevant to necrotizing enterocolitis and other dysbiotic conditions in preterm infants.</p
Moving Away from the Reference Genome: Evaluating a Peptide Sequencing Tagging Approach for Single Amino Acid Polymorphism Identifications in the Genus <i>Populus</i>
The genetic diversity across natural
populations of the model organism, <i>Populus</i>, is extensive,
containing a single nucleotide polymorphism
roughly every 200 base pairs. When deviations from the reference genome
occur in coding regions, they can impact protein sequences. Rather
than relying on a static reference database to profile protein expression,
we employed a peptide sequence tagging (PST) approach capable of decoding
the plasticity of the <i>Populus</i> proteome. Using shotgun
proteomics data from two genotypes of <i>P. trichocarpa</i>, a tag-based approach enabled the detection of 6653 unexpected sequence
variants. Through manual validation, our study investigated how the
most abundant chemical modification (methionine oxidation) could masquerade
as a sequence variant (Ala→Ser) when few site-determining ions
existed. In fact, precise localization of an oxidation site for peptides
with more than one potential placement was indeterminate for 70% of
the MS/MS spectra. We demonstrate that additional fragment ions made
available by high energy collisional dissociation enhances the robustness
of the peptide sequence tagging approach (81% of oxidation events
could be exclusively localized to a methionine). We are confident
that augmenting fragmentation processes for a PST approach will further
improve the identification of single amino acid polymorphism in <i>Populus</i> and potentially other species as well
Moving Away from the Reference Genome: Evaluating a Peptide Sequencing Tagging Approach for Single Amino Acid Polymorphism Identifications in the Genus <i>Populus</i>
The genetic diversity across natural
populations of the model organism, <i>Populus</i>, is extensive,
containing a single nucleotide polymorphism
roughly every 200 base pairs. When deviations from the reference genome
occur in coding regions, they can impact protein sequences. Rather
than relying on a static reference database to profile protein expression,
we employed a peptide sequence tagging (PST) approach capable of decoding
the plasticity of the <i>Populus</i> proteome. Using shotgun
proteomics data from two genotypes of <i>P. trichocarpa</i>, a tag-based approach enabled the detection of 6653 unexpected sequence
variants. Through manual validation, our study investigated how the
most abundant chemical modification (methionine oxidation) could masquerade
as a sequence variant (Ala→Ser) when few site-determining ions
existed. In fact, precise localization of an oxidation site for peptides
with more than one potential placement was indeterminate for 70% of
the MS/MS spectra. We demonstrate that additional fragment ions made
available by high energy collisional dissociation enhances the robustness
of the peptide sequence tagging approach (81% of oxidation events
could be exclusively localized to a methionine). We are confident
that augmenting fragmentation processes for a PST approach will further
improve the identification of single amino acid polymorphism in <i>Populus</i> and potentially other species as well
Characterizing the Range of Extracellular Protein Post-Translational Modifications in a Cellulose-Degrading Bacteria Using a Multiple Proteolyic Digestion/Peptide Fragmentation Approach
Post-translational modifications
(PTMs) are known to play a significant
role in many biological functions. The focus of this study is to optimize
an integrated experimental/informatics approach to more confidently
characterize the range of post-translational modifications of the
cellulosome protein complex used by the bacterium Clostridium
thermocellum to better understand how this protein
machine is tuned for enzymatic cellulose solubilization. To enhance
comprehensive characterization, the extracellular cellulosome proteins
were analyzed using multiple proteolytic digests (trypsin, Lys-C,
Glu-C) and multiple fragmentation techniques (collisionally activated
dissociation, electron transfer dissociation, decision tree). As expected,
peptide and protein identifications were increased by utilizing alternate
proteases and fragmentation methods, in addition to the increase in
protein sequence coverage. The complementarity of these experiments
also allowed for a global exploration of PTMs associated with the
cellulosome based upon a set of defined PTMs that included methylation,
oxidation, acetylation, phosphorylation, and signal peptide cleavage.
In these experiments, 85 modified peptides corresponding to 28 cellulosome
proteins were identified. Many of these modifications were located
in active cellulolytic or structural domains of the cellulosome proteins,
suggesting a level of possible regulatory control of protein function
in various cellulotyic conditions. The use of complementary proteolytic
digestion/peptide fragmentation processes allowed for independent
verification of PTMs in different experiments, thus leading to increased
confidence in PTM identifications
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