Unipept 4.0: Functional Analysis of Metaproteome Data

Singh, Robbert Gurdeep; Tanca, Alessandro; Palomba, Antonio; Jeugt, Felix Van der; Verschaffelt, Pieter; Uzzau, Sergio; Martens, Lennart; Dawyndt, Peter; Mesuere, Bart

doi:10.1021/acs.jproteome.8b00716

Cited by 119 publications

(98 citation statements)

References 35 publications

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“…To characterize the taxonomic and functional distribution of microbial Kac proteins, we uploaded all the 35,200 Kac peptide sequences identified in this study into Unipept for biodiversity analysis [23]. Among all the Kac peptide sequences, 28,321 peptides (80%) were assigned to the kingdom Bacteria and 24,785 peptides could be classified at phylum level (15,170 from Firmicutes, 7876 from Bacteroidetes and 1739 from other phyla; Fig.…”

Section: Resultsmentioning

confidence: 99%

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Deep characterization of the protein lysine acetylation in human gut microbiome and its alterations in patients with Crohn’s disease

Zhang

Ning

Mayne

et al. 2019

Preprint

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22Metagenomic and metaproteomic approaches have been used to study the composition 23 and functions of the microbiota. However, no studies have examined post-translational 24 modifications (PTM) on human microbiome proteins at the metaproteome level, and it 25 remains unknown whether the microbial PTM is altered or not in patient microbiome. 26 Herein we used anti-acetyl-lysine (Kac) antibody enrichment strategy and mass 27 spectrometry to characterize the protein lysine acetylation in human microbiome, which 28 successfully identified 35,200 Kac peptides corresponding to 31,821 Kac sites from the 29 microbial or host proteins in human gut microbiome samples. The gut microbial proteins 30 exhibited Kac motifs that were distinct from those of human proteins. Functional analysis 31 showed that microbial Kac proteins were significantly enriched in energy production and 32 abundant in enzymes related to transferases and oxidoreductases. Applying to the 33 analysis of pediatric Crohn's disease (CD) patient microbiome identified 52 host and 136 34 microbial protein Kac sites that were differentially abundant in CD versus controls. 35 Interestingly, most of the decreased Kac sites in CD were derived from Firmicutes and 36 most of the increased sites were derived from Bacteroidetes. Forty-six out of the 52 37 differentially abundant human protein Kac sites were increased in CD patients, including 38 those on calprotectin, lactotransferrin and immunoglobulins. Taken together, this study 39 provides an efficient approach to study the lysine acetylation in microbiome and revealed 40 taxon-specific alterations in the lysine acetylome as well as changes in host protein 41 acetylation levels in intestinal samples during the on-set of disease in CD patients. 42 43 Keywords 44 Crohn's disease, lysine acetylation, mass spectrometry, metaproteomics, microbiome 45 46 Protein name Low High Z-score

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Section: Resultsmentioning

confidence: 99%

“…Taxonomic annotation of both unmodified and Kac peptides was performed using Unipept 4.0 with the “Equal I and L” and “Advanced misscleavage handling” options allowed [23]. Gene ontology (GO) term and Enzyme Commission (EC) number annotations were directly exported from Unipept analysis (https://unipept.ugent.be/).…”

Section: Methodsmentioning

confidence: 99%

Deep characterization of the protein lysine acetylation in human gut microbiome and its alterations in patients with Crohn’s disease

Zhang

Ning

Mayne

et al. 2019

Preprint

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“…Relative bacterial abundance in co-cultures was estimated based on strain unique peptides identified with Unipept version 4.0 55 . To exclude peptides shared between closely related strains from the analyses, all peptide sequences quantified via Proteome Discoverer were imported into the Unipept web server and analyzed with the settings “Equate I and L” and “Advanced missed cleavage handling” activated.…”

Section: Methodsmentioning

confidence: 99%

Butyrate producing Clostridiales utilize distinct human milk oligosaccharides correlating to early colonization and prevalence in the human gut

Pichler

Yamada

Shuoker

et al. 2020

Preprint

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Butyrate producing Clostridiales utilize distinct human milk oligosaccharides correlating to early 2 colonization and prevalence in the human gut 3Bacterial growth studies were performed by M.J.P. Proteomic analyses were done by M.J.P and 26 E.S. Protein characterization was done by M.J.P and M.L.L. Enzymatic characterization of 27RiLe a/b 136 was performed by A.G.,To.K., M.S. and Ta.K. Mucin preparation was performed by 28 B.S. and M.J.P. Mucin glycomics were performed by B.S., C.J. and N.G.K. Protein X-ray 29 crystallography was performed by C.Y. and S.F. Metagenome analysis were performed by C.A-30 S and M.A. Experiments were designed by M.J.P and M.A.H. The manuscript was drafted by 31 M.J.P and M.A.H and finalized with contributions of all authors. 32 33 34 35 Abstract 36The early life human gut microbiota exerts life-long health effects on the host, but the mechanisms 37 underpinning its assembly remain elusive. Particularly, the early colonization of Clostridiales from 38the Roseburia-Eubacterium group, associated with protection from colorectal cancer, immune-39and metabolic disorders is enigmatic. Here we unveil the growth of Roseburia and Eubacterium 40 members on human milk oligosaccharides (HMOs) using an unprecedented catabolic apparatus. 41The described HMO pathways and additional glycan utilization loci confer co-growth with 42 Akkermansia muciniphilia via cross-feeding and access to mucin O-glycans. Strikingly, both, HMO 43and xylooligosaccharide pathways, were active simultaneously attesting an adaptation to a mixed 44HMO-solid food diet. Analyses of 4599 Roseburia genomes underscored the preponderance of 45HMO pathways and highlighted different HMO utilization phylotypes. Our revelations provide a 46 possible rationale for the early establishment and resilience of butyrate producing Clostridiales 47and expand the role of specific HMOs in the assembly of the early life microbiota. 48 49 129 R. inulinivorans, respectively. In R. inulinivorans two additional loci encoding sialic acid and 130 fucose catabolism proteins, were also upregulated ( Supplementary Fig. 3). 131 132 133 Fig. 1: Growth of R. hominis, R. inulinivorans and E. ramulus on HMOs and upregulation of core HMOs 134 utilization loci: Growth curves of R. hominis (a) and R. inulinivorans (b) on glucose, LNT, GNB, LNB, and/or purified 135 HMOs from mothers milk compared to a no-carbon source controls over 24 h. c, Growth levels of R. inulinivorans on 136 LNT, LNB, GNB and of E. ramulus on LNT within 24 h including glucose and a non-carbon source controls. d, Growth 137 of R. hominis, R. inulinivorans and E. ramulus on monosaccharides from HMOs and mucin after 24 h. The growth 138 analyses (a-d) on media supplemented with 0.5 % (w/v) carbohydrates (except for R. inulinivorans on 1% (w/v) and 139 4% (w/v) purified HMOs from mothers milk) are means of triplicates with standard deviations. e) HMO and mucin-140 derived oligo-and monosaccharides used for the growth analyses in (a-d). The core HMO utilization loci in R. hominis 141 (f) and R. in...

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“…S2 are based on the projected estimates, as the software had consumed 14 GB out of 16 GB available RAM in the first 24 hours while only processing the first 2 million (out of 140 million) protein sequences. Unipept times and RAM requirements are taken from the latest Unipept publication 1 as the Unipept team reported using a high performance computer, and additionally database creation is not a feature of the software that is available for end users to perform. For taxa annotation speed, three files of 5,000 peptides were randomly drawn from the database, and were taxonomically annotated using Unipept’s “pept2taxa” and ProteoClade’s “annotate_peptides” functions.…”

Section: Methodsmentioning

confidence: 99%

“…Several software tools provide one or more of these features, but have practical and technical limitations that render them unable to facilitate complete analysis pipelines of quantitative proteomics data and scale to the rapidly increasing number of available reference protein sequences 4,5 . With regard to annotating peptides to taxa, Unipept is a commonly used taxonomic annotation tool that can provide access to the entire UniProt sequence repository, provides web-based visualizations and a command line interface, and was demonstrated to annotate peptides orders of magnitude faster than a prior UniProt-based application, Peptide Match 6,7 . However, the Unipept database is unavailable for the end-user to customize, is restricted to a fixed set of assumed experimental parameters such as protease, cannot be used with custom protein databases such as those generated by sequencing, and lacks capability for protein quantitation which limits its utility for analyzing many experimental data sets.…”

Section: Main Textmentioning

confidence: 99%

ProteoClade: a taxonomic toolkit for multi-species and metaproteomic analysis

Mooradian

Post

Naegle

et al. 2019

Preprint

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15We present ProteoClade, a Python toolkit that performs taxa-specific peptide assignment, protein infer-16 ence, and quantitation for multi-species proteomics experiments. ProteoClade scales to hundreds of 17 millions of protein sequences, requires minimal computational resources, and is open source, multi-18 platform, and accessible to non-programmers. We demonstrate its utility for processing quantitative 19 proteomic data derived from patient-derived xenografts and its speed and scalability enable a novel de 20 novo proteomic workflow for complex microbiota samples. 21 22Main Text: 23The goal of metaproteomic and multispecies proteomic studies is to characterize the proteomes of sam-24 ples containing multiple, comingled species, which can provide insight into the complex interactions at 25 the interface between organisms. Proteomic analysis of these samples can quantify thousands of pro-26 teins from hundreds of species in a single mass spectrometry experiment 1 , characterize education of 27 stromal tissue by patient-derived xenografts (PDXs) 2 , and extensively characterize the human oral mi-28 crobiome 3 . 29Metaproteomic and multispecies data analyses depend on the ability to integrate reference protein se-30 quence databases, taxonomic lineages, in silico proteolytic digestion, peptide identification, and quanti-31 tation. These studies universally perform bottom-up analysis, where proteins are digested into peptides 32 with a protease, and therefore require assignment of peptides to proteins based on their taxonomic 33 specificity. Several software tools provide one or more of these features, but have practical and tech-34 nical limitations that render them unable to facilitate complete analysis pipelines of quantitative prote-35 omics data and scale to the rapidly increasing number of available reference protein sequences 4,5 . With 36 regard to annotating peptides to taxa, Unipept is a commonly used taxonomic annotation tool that can 37 provide access to the entire UniProt sequence repository, provides web-based visualizations and a

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Unipept 4.0: Functional Analysis of Metaproteome Data

Cited by 119 publications

References 35 publications

Deep characterization of the protein lysine acetylation in human gut microbiome and its alterations in patients with Crohn’s disease

Deep characterization of the protein lysine acetylation in human gut microbiome and its alterations in patients with Crohn’s disease

Butyrate producing Clostridiales utilize distinct human milk oligosaccharides correlating to early colonization and prevalence in the human gut

ProteoClade: a taxonomic toolkit for multi-species and metaproteomic analysis

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