SpotLink enables sensitive and precise identification of site non-specific cross-links at the proteome scale

We developed SpotLink software for identifying site non-specific cross-links at the proteome scale. Contributed by the dual pointer dynamic pruning (DPDP) algorithm and the quality control of cross-linking sites, SpotLink identified more than 3000 cross-links from human proteome database with rich site information in a few days. We demonstrated that SpotLink outperformed other approaches in terms of sensitivity and precision on a simulated dataset and a protein complexes dataset with known structures. Additionally, we discovered some valuable protein-protein interaction (PPI) information contained in the protein complexes dataset and HeLa dataset, indicating the unique identification advantages of site non-specific cross-linking. The excellent performance of SpotLink will increase the usage of site non-specific cross-linking in the near future. SpotLink is publicly available on GitHub [https://github.com/DICP1810/SpotLink].

FunOrder: A robust and semi-automated method for the identification of essential biosynthetic genes through computational molecular co-evolution

Secondary metabolites (SMs) are a vast group of compounds with different structures and properties that have been utilized as drugs, food additives, dyes, and as monomers for novel plastics. In many cases, the biosynthesis of SMs is catalysed by enzymes whose corresponding genes are co-localized in the genome in biosynthetic gene clusters (BGCs). Notably, BGCs may contain so-called gap genes, that are not involved in the biosynthesis of the SM. Current genome mining tools can identify BGCs, but they have problems with distinguishing essential genes from gap genes. This can and must be done by expensive, laborious, and time-consuming comparative genomic approaches or transcriptome analyses. In this study, we developed a method that allows semi-automated identification of essential genes in a BGC based on co-evolution analysis. To this end, the protein sequences of a BGC are blasted against a suitable proteome database. For each protein, a phylogenetic tree is created. The trees are compared by treeKO to detect co-evolution. The results of this comparison are visualized in different output formats, which are compared visually. Our results suggest that co-evolution is commonly occurring within BGCs, albeit not all, and that especially those genes that encode for enzymes of the biosynthetic pathway are co-evolutionary linked and can be identified with FunOrder. In light of the growing number of genomic data available, this will contribute to the studies of BGCs in native hosts and facilitate heterologous expression in other organisms with the aim of the discovery of novel SMs.

Leaf Proteome Response to Drought Stress and Antioxidant Potential in Tomato (Solanum lycopersicum L.)

Advances in proteome research have opened the gateway to understanding numerous metabolic pathways and fundamental mechanisms involved in abiotic stress tolerance. In the present study, the antioxidant capacity of four tomato genotypes i.e., Kashi Amrit, Kashi Anupam, EC-317-6-1, and WIR-4360 was determined under drought stress to ascertain the scavenging potential for reactive oxygen species (ROS). A significant increase in the superoxide dismutase (SOD), Ascorbate peroxidase (APX), and catalase (CAT) activities in all the four genotypes under drought stress was observed, which seemed to be associated with a protective role against ROS (p < 0.001). Based on the antioxidant enzyme activities, a proteomic approach was applied to study differential protein expression in two selected genotypes from different species i.e., EC-317-6-1 (Solanum pimpinellifolium) and Kashi Amrit (Solanum lycopersicum) grown under irrigated, drought, and re-watering conditions. To reveal the protein network regulated under these conditions, two-dimensional gel electrophoresis was employed to identify and quantify the number of proteins in drought-sensitive (Kashi Amrit) and tolerant (EC-317-6-1) genotypes. Matrix-assisted laser desorption/ionization-time of flight analysis (MALDI-TOF) revealed a total of 453 spots after fine-tuning factors i.e., smoothness, saliency, and minimum area that responded to drought. Out of 453 total spots, 93 spots were identified in Kashi Amrit and 154 in EC-317-6-1 under irrigated conditions, whereas 4 spots were identified in Kashi Amrit and 77 spots in EC-317-6-1 under drought conditions. Furthermore, differentially expressed proteins were distinguished according to the fold change of their expression. Information provided in this report will be useful for the selection of proteins or genes in analyzing or improving drought tolerance in tomato cultivars. These findings may assist in the construction of a complete proteome database encompassing various divergent species which could be a valuable source for the improvement of crops under drought-stress conditions in the future.

Assessment of a complete and classified platelet proteome from genome-wide transcripts of human platelets and megakaryocytes covering platelet functions

Novel platelet and megakaryocyte transcriptome analysis allows prediction of the full or theoretical proteome of a representative human platelet. Here, we integrated the established platelet proteomes from six cohorts of healthy subjects, encompassing 5.2 k proteins, with two novel genome-wide transcriptomes (57.8 k mRNAs). For 14.8 k protein-coding transcripts, we assigned the proteins to 21 UniProt-based classes, based on their preferential intracellular localization and presumed function. This classified transcriptome-proteome profile of platelets revealed: (i) Absence of 37.2 k genome-wide transcripts. (ii) High quantitative similarity of platelet and megakaryocyte transcriptomes (R = 0.75) for 14.8 k protein-coding genes, but not for 3.8 k RNA genes or 1.9 k pseudogenes (R = 0.43–0.54), suggesting redistribution of mRNAs upon platelet shedding from megakaryocytes. (iii) Copy numbers of 3.5 k proteins that were restricted in size by the corresponding transcript levels (iv) Near complete coverage of identified proteins in the relevant transcriptome (log2fpkm > 0.20) except for plasma-derived secretory proteins, pointing to adhesion and uptake of such proteins. (v) Underrepresentation in the identified proteome of nuclear-related, membrane and signaling proteins, as well proteins with low-level transcripts. We then constructed a prediction model, based on protein function, transcript level and (peri)nuclear localization, and calculated the achievable proteome at ~ 10 k proteins. Model validation identified 1.0 k additional proteins in the predicted classes. Network and database analysis revealed the presence of 2.4 k proteins with a possible role in thrombosis and hemostasis, and 138 proteins linked to platelet-related disorders. This genome-wide platelet transcriptome and (non)identified proteome database thus provides a scaffold for discovering the roles of unknown platelet proteins in health and disease.

Proteomic Identification of Bombyx mori Organelles Using the Engineered Ascorbate Peroxidase APEX and Development of Silkworm Organelle Proteome Database (SilkOrganPDB)

Silkworm Bombyx mori is an economically important insect and a lepidopteran model. Organelle proteome is vital to understanding gene functions; however, it remains to be identified in silkworm. Here, using the engineered ascorbate peroxidase APEX, we constructed transgenic B. mori embryo cells (BmE) expressing APEX-NLS, COX4-APEX, APEX-Rev, and APEX-KDEL in nucleus, mitochondrial matrix (MM), cytosol, and endoplasmic reticulum (ER), and isolated the biotin-labeled proteins using streptavidin-affinity purification, respectively. The isolated proteins were determined using LC-MS/MS and annotated by searching B. mori genomes downloaded from GenBank, SilkBase, SilkDB 2.0, and SilkDB 3.0, resulting in 842, 495, 311, and 445 organelle proteins identified, respectively. We mapped the 296 MM proteins annotated in the GenBank data to mitochondrial protein databases of the fly, human, and mouse, and found that 140 (47%) proteins are homologous to 80 fly proteins, and 65 (22%) proteins match to 31 and 29 human and mouse proteins, respectively. Protein orthology was predicted in multiple insects using OrthoMCL, producing 460 families containing 839 proteins we identified. Out of 460 families, 363 were highly conserved and found in all insects, leaving only three proteins without orthology in other insects, indicating that the identified proteins are highly conserved and probably play important roles in insects. A gene ontology enrichment analysis by clusterProfiler revealed that the nucleus proteins significantly enriched in cellular component terms of nucleus and nucleolus, the MM proteins markedly enriched in molecular function terms of nucleotide binding, and the cytosol proteins mainly enriched in biological process terms of small molecule metabolism. To facilitate the usage and analysis of our data, we developed an open-access database, Silkworm Organelle Proteome Database (SilkOrganPDB), which provides multiple modules for searching, browsing, downloading, and analyzing these proteins, including BLAST, HMMER, Organelle Proteins, Protein Locations, Sequences, Gene Ontology, Homologs, and Phylogeny. In summary, our work revealed the protein composition of silkworm BmE organelles and provided a database resource helpful for understanding the functions and evolution of these proteins.

A Comprehensive Urine Proteome Database Generated From Patients With Various Renal Conditions and Prostate Cancer

Urine proteins can serve as viable biomarkers for diagnosing and monitoring various diseases. A comprehensive urine proteome database, generated from a variety of urine samples with different disease conditions, can serve as a reference resource for facilitating discovery of potential urine protein biomarkers. Herein, we present a urine proteome database generated from multiple datasets using 2D LC-MS/MS proteome profiling of urine samples from healthy individuals (HI), renal transplant patients with acute rejection (AR) and stable graft (STA), patients with non-specific proteinuria (NS), and patients with prostate cancer (PC). A total of ~28,000 unique peptides spanning ~2,200 unique proteins were identified with a false discovery rate of &lt;0.5% at the protein level. Over one third of the annotated proteins were plasma membrane proteins and another one third were extracellular proteins according to gene ontology analysis. Ingenuity Pathway Analysis of these proteins revealed 349 potential biomarkers in the literature-curated database. Forty-three percentage of all known cluster of differentiation (CD) proteins were identified in the various human urine samples. Interestingly, following comparisons with five recently published urine proteome profiling studies, which applied similar approaches, there are still ~400 proteins which are unique to this current study. These may represent potential disease-associated proteins. Among them, several proteins such as serpin B3, renin receptor, and periostin have been reported as pathological markers for renal failure and prostate cancer, respectively. Taken together, our data should provide valuable information for future discovery and validation studies of urine protein biomarkers for various diseases.

Comparative proteomic profiling of newly acquired, virulent and attenuated Neoparamoeba perurans proteins associated with amoebic gill disease

The causative agent of amoebic gill disease, Neoparamoeba perurans is reported to lose virulence during prolonged in vitro maintenance. In this study, the impact of prolonged culture on N. perurans virulence and its proteome was investigated. Two isolates, attenuated and virulent, had their virulence assessed in an experimental trial using Atlantic salmon smolts and their bacterial community composition was evaluated by 16S rRNA Illumina MiSeq sequencing. Soluble proteins were isolated from three isolates: a newly acquired, virulent and attenuated N. perurans culture. Proteins were analysed using two-dimensional electrophoresis coupled with liquid chromatography tandem mass spectrometry (LC–MS/MS). The challenge trial using naïve smolts confirmed a loss in virulence in the attenuated N. perurans culture. A greater diversity of bacterial communities was found in the microbiome of the virulent isolate in contrast to a reduction in microbial community richness in the attenuated microbiome. A collated proteome database of N. perurans, Amoebozoa and four bacterial genera resulted in 24 proteins differentially expressed between the three cultures. The present LC–MS/MS results indicate protein synthesis, oxidative stress and immunomodulation are upregulated in a newly acquired N. perurans culture and future studies may exploit these protein identifications for therapeutic purposes in infected farmed fish.

A comprehensive urine proteome database generated from patients with various renal conditions and prostate cancer

Urine proteins can serve as viable biomarkers for diagnosing and monitoring various diseases. A comprehensive urine proteome database, generated from a variety of urine samples with different disease conditions, can serve as a reference resource for facilitating discovery of potential urine protein biomarkers. Herein, we present a urine proteome database generated from multiple datasets using 2D LC-MS/MS proteome profiling of urine samples from healthy individuals (HI), renal transplant patients with acute rejection (AR) and stable graft (STA), patients with non-specific proteinuria (NS), and patients with prostate cancer (PC). A total of ~28,000 unique peptides spanning ~2,200 unique proteins were identified with a false discovery rate of <0.5% at the protein level. Over one third of the annotated proteins were plasma membrane proteins and another one third were extracellular proteins according to gene ontology analysis. Ingenuity Pathway Analysis of these proteins revealed 349 potential biomarkers. Surprisingly, 43% (167) of all known cluster of differentiation (CD) proteins were identified in the various human urine samples. Interestingly, following comparisons with five recently published urine proteome profiling studies, which applied similar approaches, there are still ~400 proteins which are unique to this current study. These may represent potential disease-associated proteins. Among them, several proteins such as myoglobin, serpin B3, renin receptor, and periostin have been reported as pathological markers for renal failure and prostate cancer, respectively. Taken together, our data should provide valuable information for future discovery and validation studies of urine protein biomarkers for various diseases.

Knock out of Specific Maternal Vitellogenins in Zebrafish (Danio Rerio) Evokes Vital Changes in Egg Proteomic Profiles that resemble the Phenotype of Poor Quality Eggs

Background We previously reported the results of CRISPR/Cas9 knock-out (KO) of type-I and type-III vitellogenins (Vtgs) in zebrafish, which provided the first experimental evidence of essentiality and disparate functioning of specific types of Vtg at different times during vertebrate development. However, the lack of knowledge on specific contributions of different types of Vtg to molecular functions related to major developmental processes remained to be investigated. The present study employed liquid chromatography and tandem mass spectrometry (LC-MS/MS) to observe proteomic profiles of zebrafish eggs lacking three type-I Vtgs (Vtg 1, 4 and 5) simultaneously (vtg1-KO), or lacking type III Vtg (vtg3) only (vtg3-KO), as compared to those of wild type (Wt) eggs. Obtained spectra were searched against a zebrafish proteome database and identified proteins were quantified based on normalized spectral counts.Results The vtg-KO in zebrafish revealed impaired proteomes of 1 hour post fertilization (hpf) zebrafish eggs which were highly resembled the proteomic phenotype of poor quality eggs of the same developmental stage reported in our prior studies. Proteomic profiles of vtg-KO eggs and perturbations in abundances of hundreds of proteins revealed unique, noncompensable contributions of multiple Vtgs in protein homeostasis (synthesis and degradation) and in energy homeostasis even after zygotic genome activation. Increase in endoplasmic reticulum stress, Redox/Detox activities, glycolysis/gluconeogenesis, enrichment in cellular proliferation and human neurodegenerative disease related mechanisms in both vtg1- and vtg3-KO eggs point to severe molecular changes and consecutive dysfunctions in the abovementioned cellular activities. Distinctive increase in apoptosis and Parkinson disease pathways and decrease in lipid metabolism related activities in vtg3-KO eggs implies compelling roles of Vtg3, the least abundant form of Vtgs in vertebrate eggs, in mitochondrial activities. Several differentially abundant proteins representing the altered molecular mechanisms were unveiled to be considered as strong candidate markers to study details of these mechanisms during early embryonic development in zebrafish and potentially other vertebrates. Conclusions These findings indicate that the global egg proteome is subject to extensive modification depending on the presence or absence of specific Vtgs and that these modifications can have a major impact on developmental competence.

A high-resolution brain proteome map uncovers the Inter-hemispheric laterality & Inter-regional protein expression changes

The human brain has always been a black box full of mysteries. Here we present one of the most comprehensive proteomics investigation of the brain, focusing on inter-hemispheric differences. An extensive mass spectrometry-based analysis of 19 brain regions from both left and right hemispheres measured more than 3300 proteins and 38700 peptides. This high-resolution data provides a comprehensive coverage of experimentally measured (non-hypothetical) proteins across various regions to characterize inter-hemispheric differences. We also tried to understand the brain proteins in terms of synapse analysis. The study has attempted to investigate the expression of neuroanatomical allied region and brain disorder protein markers in 19 region and sub-region of brain. Furthermore, we have developed the most comprehensive Brain Proteome Database, based on our, and publicly available curated data representing more than 9000 proteins (with isoforms) and around 90000 peptides at www.brainprot.org, which can aid in understanding the human brain’s complexity.