Protein Identification Technology Research Articles

Retrieve and Revise: Improving Peptide Identification with Similar Mass Spectra

Tandem mass spectrometry is an indispensable technology for identification of proteins from complex mixtures. Accurate and sensitive analysis of large amounts of mass spectra data is a principal challenge in proteomics. Conventional deep learning-based peptide identification models usually adopt an encoder-decoder framework and generate target sequence from left to right without fully exploiting the global information. A few recent approaches seek to employ two-pass decoding, yet have limitations when facing the spectra filled with noise. In this paper, we propose a new paradigm for improved peptide identification, which first retrieves a similar mass spectrum from the database as a reference and then revise the matched sequence according to the difference information between the referenced spectrum and current context. The inspiration of design comes that the retrieved peptide-spectrum pair provides a good start point and indirect access to both past and future information, such that each revised amino acid can be produced with better noise perception and global understanding. Moreover, a disturb-based optimization process is introduced to sharpen the attention for difference vector with reinforcement learning before fed to decoder. Experimental results on several public datasets demonstrate that prominent performance boost is obtained with the proposed method. Remarkably, we achieve new state-of-the-art identification results on these datasets.

Multiple Mechanisms of DNA Methylation in Cancer Initiation and Development

Epigenetic refers to DNA methylation, histone modification and histone variants, to change expression level of heritable molecular determinants without the changing DNA sequence. With the development of the sequencing and protein identification technology, a large number of studies from International Cancer Genome Consortium(ICGC) illustrates Epigenetic alterations play an important part in cancer initiation and metastasis. The proteomic and genomic studies find out the potential connection and mechanism between epigenetics and cancers. Nowadays, the epigenetic inhibitors are still the main therapies in clinical treatments. This article aims to find out the relationship between DNA methylation and cancer, and create a new version, where epigenetic alterations combines cancer cells to open a new gate of organ transplantation. In conclusion, using DNA methylation to transform the cancer cells into specific cells to build a new organ may solve the most common problem, cell pairings, in organ transplantation.

Proteomics analysis of Trichoplusia ni midgut epithelial cell brush border membrane vesicles.

The insect midgut epithelium is composed of columnar, goblet, and regenerative cells. Columnar epithelial cells are the most abundant and have membrane protrusions that form the brush border membrane (BBM) on their apical side. These increase surface area available for the transport of nutrients, but also provide opportunities for interaction with xenobiotics such as pathogens, toxins and host plant allelochemicals. Recent improvements in proteomic and bioinformatics tools provided an opportunity to determine the proteome of the T. ni BBM in unprecedented detail. This study reports the identification of proteins from BBM vesicles (BBMVs) using single dimension polyacrylamide gel electrophoresis coupled with multi‐dimensional protein identification technology. More than 3000 proteins were associated with the BBMV, of which 697 were predicted to possess either a signal peptide, at least one transmembrane domain or a GPI‐anchor signal. Of these, bioinformatics analysis and manual curation predicted that 185 may be associated with the BBMV or epithelial cell plasma membrane. These are discussed with respect to their predicted functions, namely digestion, nutrient uptake, cell signaling, development, cell–cell interactions, and other functions. We believe this to be the most detailed proteomic analysis of the lepidopteran midgut epithelium membrane to date, which will provide information to better understand the biochemical, physiological and pathological processes taking place in the larval midgut.

Bioinformatic and Functional Analysis of Venom from the Jewel Wasp Ampulex compressa

The parasitoid jewel wasp Ampulex compressa exploits its host, the American cockroach Periplaneta americana, as a food source for its young. The host is subdued through direct envenomation into the central nervous system, inducing a days‐long “zombie‐like state” referred to as hypokinesia. Hypokinesia is a specific, venom‐induced behavioral state characterized by suppression of the escape response and reduced spontaneous walking, leaving other motor functions intact. This condition is reversible, provided that egg deposition is prevented. To facilitate elucidation of biochemical mechanisms underlying venom‐induced hypokinesia, we developed a comprehensive inventory of the venom by combining: 1) next‐generation RNA sequencing, transcriptomic analysis of venom gland components, and 2) proteomics of venom apparatus extracts and whole milked venom via mass spectroscopy‐based Multi‐Dimensional Protein Identification Technology. These analyses yielded a total of 201 proteins and peptides in the venom, 105 with Pfam domains, 146 similar to other proteins in the NCBI‐nr or Uniprot databases, and 45 novel proteins absent in global databases. A. compressa venom proteins include members of the M13 family of proteases (11), phospholipase A2 isoforms (10), along with hyaluronidase, chitinase, icarapin, venom acid phosphatase, serpins, and signaling peptides including tachykinin, corazonin, and myosuppressin. Differential expression analysis and proteomics of the two major tissues of the venom apparatus ‐ venom sac and venom gland ‐identified tissue‐specific expression of key venom components.We show that A. compressa venom tachykinins (AcVTK) are able to activate the cockroach brain tachykinin receptor with high affinity (EC50 ~1 nM) in vitro. Injection of synthetic AcVTK into the subesophageal ganglion, an established target of the wasp venom, increases escape response threshold for one hour post‐treatment. These results indicate that tachykinin may participate in the behavioral changes resulting from envenomation of cockroach cerebral ganglia.We isolated and characterized two novel, paralogous venom sac peptides found in milked venom. These peptides are alpha‐helical, amphipathic, and constitute a new family of venom toxins termed ampulexins. Injection of the most abundant venom peptide, ampulexin‐1, into the subesophageal ganglion of cockroaches resulted in a short‐term increase in escape threshold.Our findings show that the venom of A. compressa is a rich collection of both well‐known and novel, pharmacologically diverse compounds. Our study provides new and comprehensive information on A. compressa venom contents that inform hypotheses regarding its mode of action.

Developing the venome of the parasitoid wasp Ampulex compressa (778.4)

The endoparasitoid wasp Ampulex compressa injects venom directly into the central nervous system of its host, the American cockroach (Periplaneta americana), to induce a long‐term behavioral state known as hypokinesia. Hypokinesia is a specific, venom‐induced behavioral state characterized by suppression of the escape response and reduced spontaneous walking, leaving other motor functions unaffected. To facilitate elucidation of the biochemical mechanism behind venom‐induced hypokinesia, we are developing a bioinformatic venome by combining next‐generation RNA sequencing and transcriptome analysis of venom gland tissue and mass spectroscopy‐based Multi‐Dimensional Protein Identification Technology (MuDPIT) from extracts of whole milked venom. Preliminary analysis has yielded identification of more than 50 proteins and peptides, with putative identifications of venom components including endothelin‐converting enzyme‐like peptidase, icarapin, venom acid phosphatase, and tachykinin along with ~20 novel peptides and proteins. The action of the venom appears to be non‐cytolytic, specific, and reversible. Venome analysis of A. compressa may yield a rich collection of novel, pharmaceutically relevant compounds.

De novo derivation of proteomes from transcriptomes for transcript and protein identification

Identification of proteins by tandem mass spectrometry requires a database of the proteins that could be in the sample. This is available for model species (e.g. humans) but not for non-model species. Ideally, for a non-model species the sequencing of expressed mRNA would generate a protein database for mass spectrometry based identification, allowing detection of genes and proteins using high throughput sequencing and protein identification technologies. Here we use human cells infected with human adenovirus as a complex and dynamic model to demonstrate this approach is robust. Our Proteomics Informed by Transcriptomics technique identifies >99% of over 3700 distinct proteins identified using traditional analysis reliant on comprehensive human and adenovirus protein lists. This facilitates high throughput acquisition of direct evidence for transcripts and proteins in non-model species. Critically, we show this approach can also be used to highlight genes and proteins undergoing dynamic changes in post transcriptional protein stability.

Efficiency of laccase production in a 65-L air-lift reactor for potential green industrial and environmental application

Abstract Laccase is one of the most useful green enzymes for cleaner industrial application and environmental protection. It is necessary to produce laccase with known composition of its commercial style at low production cost in an efficient way. In this study, the efficiency of laccase production by Pycnoporus sp. SYBC-L3 in a 65-L air-lift reactor was evaluated and the main extracellular proteins were identified. The highest laccase activity approximated 72,000 U/L after 6 days' cultivation. Using nano-LC–MS/MS protein identification technology, 22 different peptide fragments were identified matching sequences in different proteins with laccase as the predominant extracellular one. The crude laccase exhibited high catalytic activity at temperatures ranging from 0 °C to 100 °C at an acidic condition, with respective relative activity of 40% at 0 °C and 80% at 100 °C. The crude laccase was found to be very thermal stable, with half-lives of 4 h, 6.5 h and 10 h at 70 °C, 60 °C and 50 °C, respectively. The crude laccase still retained approximately 85% of its initial activity after one year of storage at room temperature. These results showed that laccase can be effectively produced by Pycnoporus sp. SYBC-L3 in air-lift reactor at larger scale and has great potential for industrial production and applications.

Proteomic characterization of the Pseudomonas putida KT2440 global response to a monocyclic aromatic compound by iTRAQ analysis and 1DE-MudPIT

Pseudomonas putida KT2440 is a metabolically versatile soil bacterium. To examine the effects of an aromatic compound on the proteome of this bacterium, cytosolic proteins induced by the presence of benzoate and succinate were analyzed using two liquid chromatography (LC)-based proteomic approaches: an isobaric tag for relative and absolute quantitation (iTRAQ) for quantitative analysis and one-dimensional gel electrophoresis/multidimensional protein identification technology (1-DE MudPIT) for protein identification. In total, 1286 proteins were identified by 1-DE MudPIT; this represents around 23.3% of the total proteome. In contrast, 570 proteins were identified and quantified by iTRAQ analysis. Of these, 55 and 52 proteins were up- and down-regulated, respectively, in the presence of benzoate. The proteins up-regulated included benzoate degradation enzymes, chemotaxis-related proteins, and ABC transporters. Enzymes related to nitrogen metabolism and pyruvate metabolism were down-regulated. These data suggest that a combination of 1-DE MudPIT and iTRAQ is an appropriate method for comprehensive proteomic analysis of biodegradative bacteria.

Resveratrol suppresses human colon cancer cell proliferation and induces apoptosis via targeting the pentose phosphate and the talin-FAK signaling pathways-A proteomic approach

BackgroundWe and others have previously reported that resveratrol (RSV) suppresses colon cancer cell proliferation and elevates apoptosis in vitro and/or in vivo, however molecular mechanisms are not fully elucidated. Particularly, little information is available on RSV's effects on metabolic pathways and the cell-extra cellular matrix (ECM) communication that are critical for cancer cell growth. To identify important targets of RSV, we analyzed whole protein fractions from HT-29 advanced human colon cancer cell line treated with solvent control, IGF-1 (10 nM) and RSV (150 μM) using LC/MS/MS-Mud PIT (Multidimensional Protein Identification Technology).ResultsPentose phosphate pathway (PPP), a vital metabolic pathway for cell cycle progression, was elevated and suppressed by IGF-1 and RSV, respectively in the HT-29 cell line. Enzymatic assays confirmed RSV suppression of glucose-6 phosphate dehydrogenase (rate limiting) and transketolase, key enzymes of the PPP. RSV (150 μM) suppressed, whereas IGF-1 (10 nM) elevated focal adhesion complex (FAC) proteins, talin and pFAK, critical for the cell-ECM communication. Western blotting analyses confirmed the suppression or elevation of these proteins in HT-29 cancer cells treated with RSV or IGF-1, respectively.ConclusionsProteomic analysis enabled us to establish PPP and the talin-pFAK as targets of RSV which suppress cancer cell proliferation and induce apoptosis in the colon cancer cell line HT-29. RSV (150 μM) suppressed these pathways in the presence and absence of IGF-1, suggesting its role as a chemo-preventive agent even in obese condition.

ITRAQ Proteomics Approaches Reveal Novel Mitochondrial Targets of Estrogen Deficiency in Aged Female Rat Heart

BackgroundThe incidence of MI rises sharply at the time of menopause, implicating loss of estrogen (E2) in age‐related increases in ischemic injury.PurposeTo identify new mitochondrial protein targets for the treatment of acute MI in aged women using a state‐of‐the‐art proteomics approach. We hypothesized that E2 deficiency exacerbates age‐dependent disruptions in mitochondrial proteins in female rat heart.MethodsTargeted proteomic analyses were conducted in mitochondria isolated from left ventricles of adult (6 mo) and aged (24 mo) F344 ovary‐intact or ovariectomized (OVX) rats using the newly released isobaric tags for relative and absolute quantification (iTRAQ) 8plex labeling and mass spectrometry (n=4‐5/group).ResultsInitial multi‐dimensional protein identification technology (MudPIT) analyses revealed n=638 distinct mitochondrial proteins. With iTRAQ, significant group differences (false discovery rate < 5%) were observed in proteins related to cellular metabolism, ion transport, oxidative stress and cell death regulation. Effects of age‐associated E2 deficiency were primarily limited to proteins which regulate oxidative stress, and were not observed in adult OVX.ConclusionsProteomic analysis through iTRAQ is effective in identifying new candidate proteins for further in‐depth study of age‐ and E2‐dependent alterations in mitochondrial cardioprotective signaling.

Modeling peptide fragmentation with dynamic Bayesian networks for peptide identification

Motivation: Tandem mass spectrometry (MS/MS) is an indispensable technology for identification of proteins from complex mixtures. Proteins are digested to peptides that are then identified by their fragmentation patterns in the mass spectrometer. Thus, at its core, MS/MS protein identification relies on the relative predictability of peptide fragmentation. Unfortunately, peptide fragmentation is complex and not fully understood, and what is understood is not always exploited by peptide identification algorithms.Results: We use a hybrid dynamic Bayesian network (DBN)/support vector machine (SVM) approach to address these two problems. We train a set of DBNs on high-confidence peptide-spectrum matches. These DBNs, known collectively as Riptide, comprise a probabilistic model of peptide fragmentation chemistry. Examination of the distributions learned by Riptide allows identification of new trends, such as prevalent a-ion fragmentation at peptide cleavage sites C-term to hydrophobic residues. In addition, Riptide can be used to produce likelihood scores that indicate whether a given peptide-spectrum match is correct. A vector of such scores is evaluated by an SVM, which produces a final score to be used in peptide identification. Using Riptide in this way yields improved discrimination when compared to other state-of-the-art MS/MS identification algorithms, increasing the number of positive identifications by as much as 12% at a 1% false discovery rate.Availability: Python and C source code are available upon request from the authors. The curated training sets are available at http://noble.gs.washington.edu/proj/intense/. The Graphical Model Tool Kit (GMTK) is freely available at http://ssli.ee.washington.edu/bilmes/gmtk.Contact:noble@gs.washington.edu

Identification of Dynamic Proteome Changes Upon Ligand Activation of Trk-Receptors Using Two-dimensional Fluorescence Difference Gel Electrophoresis and Mass Spectrometry

The TrkA and TrkB tyrosine kinases are members of the neurotrophin receptor family and mediate survival, differentiation, growth, and apoptosis of neurons in response to stimulation by their ligands, NGF and BDNF, respectively. Expression levels of TrkA/TrkB are important prognostic factors in a variety of embryonal tumors including neuroblastoma, the most common solid tumor of childhood. Because TrkA/TrkB exhibit a high level of sequence similarity and use overlapping pathways for signal transduction, the existence of specific effector molecules crucial for receptor and cell-type-specific response is likely. To identify these effectors by analyzing biological effects of TrkA and TrkB activation in a defined model, we performed a proteome study using the human neuroblastoma SY5Y cell line stably transfected with the TrkA or TrkB cDNA. The use of the recently introduced DIGE (fluorescence two-dimensional difference gel electrophoresis) system (Amersham Biosciences, Piscataway, NJ) allowed us to monitor differences in protein expression between samples in one gel. Proteomic changes were monitored in a time course of 0, 0.5, 1, 6, and 24 h following receptor activation. Using MALDI mass spectrometry, we identified, respectively, 22 and 9 differentially expressed proteins upon the addition of neurotrophin in SY5Y-TrkB and SY5Y-TrkA cells. Functional assignment revealed that the majority of these proteins are involved in organization and maintenance of cellular structures.

Ultrasensitive detection of unstained proteins in acrylamide gels by native UV fluorescence.

Visualization of proteins inside acrylamide and other gels usually relies on different staining methods. To omit the protein-staining procedure, we visualized unstained proteins inside acrylamide gels by laser excitation with ultraviolet (UV) light (280 nm, 35 mJ/cm2) and directly detected native UV fluorescence. In one-dimensional gels, a detection limit as low as 1 ng for bovine serum albumin and 5 ng for other proteins with a linear dynamic range (2.7 orders of magnitude) comparable to state of the art fluorescent dyes could be achieved. In addition, the application of this method to 20 microg of a whole cell lysate separated in a two-dimensional gel showed more than 600 spots. Since protein labeling always represents a serious obstacle in protein identification technologies, the working efficiency with our procedure can be considered as a significant improvement for protein visualization and reproducibility in proteomics.