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