Proteomics

High-throughput analysis of peptide-binding modules

by Bernard Liu

Application of Genomic and Proteomic Technologies in Biomarker Discovery

by Elana Fertig

EJ Fertig, R Slebos, and CH Chung. Application of Genomic and Proteomic Technologies in Biomarker Discovery. In: Govindan R, ed. 2012 ASCO Educational Book. Alexandria, VA: American Society of Clinical Oncology; 2012;377-382.

Overview: Sequencing of the human genome was completed in 2001. Building on the technology and experience of... more Overview: Sequencing of the human genome was completed in 2001. Building on the technology and experience of whole-exome sequencing, numerous cancer genomes have been sequenced, including head and neck squamous cell carcinoma (HNSCC) in 2011. Although DNA sequencing data reveals a complex genome with numerous mutations, the biologic interaction and clinical significance of the overall genetic aberrations are largely unknown. Comprehensive analyses of the tumors using genomics and proteomics beyond sequencing data can potentially accelerate the rate and number of biomarker discoveries to improve biology-driven classification of tumors for prognosis and patient selection for a specific therapy. In this review, we will summarize the current genomic and proteomic technologies, general biomarker-discovery paradigms using the technology and published data in HNSCC---including potential clinical applications and limitations.

Proteomic Profiling of the Photo-oxidation of Silk Fibroin: Implications for Historic Tin-Weighted Silk

by Caroline Solazzo

The stability of silk proteins to ultraviolet light is an issue of significant concern in both the appearance... more The stability of silk proteins to ultraviolet light is an issue of significant concern in both the appearance retention of silk-derived products and the preservation of historic silk textiles. Until now, evaluation of silk degradation has only been performed at the holistic, rather than molecular level. This paper describes the first proteomic profiling of silk photo-oxidation, characterising protein primary level modification leading to coloration changes, and evaluating the effects of tin weighting on photodegradation. Heavy chain fibroin, the main proteinaceous component of the silk thread, is a repetitive, highly crystalline protein with a content rich in tyrosine. Photoproducts of tyrosine were characterised and the levels of oxidative modification at the protein primary structural level correlated with changes in coloration and tensile strength. The effect of tin as a weighting agent used on historical fabrics was examined. Tin-weighted fabrics were evaluated following two treatments (pink and dynamite) and proteomic analysis revealed a significant increase in oxidatively modified amino acid residues within the pink treated silk. These findings offer significant new insight into the molecular-level oxidation of silk proteins under UV exposure, and the effects of silk treatments in either exacerbating or ameliorating this degradation.

Proteogenomic analysis of Mycobacterium tuberculosis by high resolution mass spectrometry

by Amit Yadav

A systematic analysis of eluted fraction of plasma post immunoaffinity depletion: implications in biomarker discovery

by Amit Yadav

MassWiz: a novel scoring algorithm with target-decoy based analysis pipeline for tandem mass spectrometry

by Amit Yadav

Renal Cyst Formation in Fh1-Deficient Mice Is Independent of the Hif/Phd Pathway: Roles for Fumarate in KEAP1 Succination and Nrf2 Signaling

by Nicola Ternette

Comparative evaluation of label‐free SINQ normalized spectral index quantitation in the central proteomics facilities pipeline

by Nicola Ternette

Discrimination of freshwater fish species by Matrix-Assisted Laser Desorption/Ionization- Time Of Flight Mass Spectrometry (MALDI-TOF MS): a pilot study.

by pietro volta

Volta P., N. Riccardi, R. Lauceri & M. Tonolla. 2012. Journal of Limnology 71: 164-169

In this study we discriminate three freshwater fish species (the shad Alosa agone Scopoli 1786, the whitefish... more In this study we discriminate three freshwater fish species (the shad Alosa agone Scopoli 1786, the whitefish Coregonus macrophthalmus Nüsslin1882 and the roach Rutilus rutilus Linnaeus 1758) by Matrix-Assisted Laser Desorption/Ionization- Time Of Flight Mass Spectrometry (MALDI-TOF MS) using both muscle and liver tissues. The technology enables to analyze tissues after a simple single-step extraction procedure without any further purification. The molecular profile of muscle tissues showed the most intense peaks at m/z range of 11,354.0 (±2.0 SD) Da, 3508.5 (±1.5 SD) Da and 8567.2 (±1.1 SD) Da for the shad, the whitefish and the roach respectively.
The molecular profiles of liver tissues exhibit most of the highest peak intensities in the range between 2000 and 6000 m/z values.
The roach shows the clearest pattern with high intensities detected at mass ranges between 3000 and 3550 Da with maxima at m/z 3035.2 (±0.2) Da and 3468.7 (±0.3) Da. The shad shows a shared high peak at m/z 3429.0 (±0.3) Da. The whitefish shows a group of major peaks in the m/z range of 3000-3700 Da with the highest being at 3635 (±0.3) Da. The overall signal pattern generated is highly specific for each species and, according to cluster analyses based on the total number of peaks, we could discriminate the three species.

Keratins in oral cancer: Necessity of mass spectrometry for validation of antibody based identifications

by Amit Fulzele

Peptide Identification by Searching Large-Scale Tandem Mass Spectra against Large Databases: Bioinformatics Methods in Proteogenomics

by Mohamed Helmy

Mohamed Helmy, Masaru Tomita, Yasushi Ishihama

Mass spectrometry-based shotgun proteomics approaches are currently considered as the technology-of-choice for... more Mass spectrometry-based shotgun proteomics approaches are currently considered as the technology-of-choice for large-scale proteogenomics due to high throughput, good availability and relative ease of use. Protein mixtures are firstly digested with protease, e. g. trypsin, and the resultant peptides are analyzed using liquid chromatography - tandem mass spectrometry. Proteins and peptides are identified from the resultant tandem mass spectra by de novo interpretation of the spectra or by searching databases of putative sequences. Since this data represents the expressed proteins in the sample, it can be used to infer novel proteogenomic features when mapped to the genome. However, high-throughput mass spectrometry instruments can readily generate hundreds of thousands, perhaps millions, of spectra and the size of genomic databases, such as six-frame translated genome databases, is enormous. Therefore, computational demands are very high, and there is potential inaccuracy in peptide identification due to the large search space. These issues are considered the main challenges that limit the utilization of this approach. In this review, we highlight the efforts of the proteomics and bioinformatics communities to develop methods, algorithms and software tools that facilitate peptide sequence identification from databases in large-scale proteogenomic studies.

The proteomes of Sydney rock oysters vary spatially according to exposure to acid sulfate runoff

by Valter Amaral

Amaral V, Thompson EL, Bishop MJ, Raftos DA, 2012 Mar Freshwat Res 63: 361-369. doi:10.1071/MF11213 [featured in the cover of the issue]

Runoff from acid sulfate soils (ASS) has large negative environmental and economic impacts on estuarine ecosystems.... more Runoff from acid sulfate soils (ASS) has large negative environmental and economic impacts on estuarine ecosystems. Oysters display reduced abundance, growth rate and shell thickness when exposed to ASS runoff, yet the molecular underpinnings of their responses have not been explored. We hypothesized that the proteomes of wild Sydney rock oysters, Saccostrea glomerata, would differ between populations that are recurrently exposed to and that are unaffected by runoff from ASS. We used two-dimensional electrophoresis to compare protein abundances in the gills of S. glomerata collected from two sites close to (acidified) and two sites away from (reference) major ASS outflow drains in a south-east Australian estuary. Approximately 5 % of the proteome was differentially expressed between oysters from acidified and reference sites, with 5 protein spots more abundant and one less abundant at the sites close to drains. Another protein spot was present only in oysters from reference sites. This study has provided the first screening of spatial variation in the protein expression of S. glomerata with respect to discharge from ASS. Altered protein expression may underpin short-term inducible responses to ASS runoff, or genetic resistance acquired through recurrent exposure of populations to the stressor.

Identification of protein remains in archaeological potsherds by proteomics

by Caroline Solazzo

We demonstrate here the possibility of identifying proteins trapped in few milligrams of the clay matrix of a... more We demonstrate here the possibility of identifying proteins trapped in few milligrams of the clay matrix of a 1200−1400 AD Iñupiat potsherd fragment from Point Barrow, Alaska, by a dedicated proteomics approach. The four main steps of a proteomics analysis, (i) protein extraction from biological samples, (ii) protein hydrolysis using a hydrolase enzyme, (iii) nanoLC, nanoESI MS, and MS/MS analysis of the generated peptides, and (iv) protein identification using protein databank proceeded from genomic data, have been optimized for archeological remains. Briefly our procedure starts by grinding the potsherds, extraction with 1% trifluoroacetic acid, digestion with excess of trypsin, nanoLC, nanoESI FT-ICR analysis, and data mining by homology search. The developed conditions were evaluated on protein extracts from remains obtained by heated muscle tissues and blubbers of different seal and whale species, these samples representing the main diet sources of the Eskimo population. Most of the proteins were identified by sequence homology to other species due to the lack of cetacean and pinniped proteins in the databanks. More interestingly, two proteins, myoglobin and hemoglobin, respectively, identified in muscle tissue samples and blubber samples highlight several specific peptides of cetacean and pinniped species; these peptides are significant to prove the presence of these marine species in the analyzed samples. Based on the developed methodology and on protein identification results obtained from the heated seal/whale muscle tissues and blubbers, the analysis of the clay matrix of a 1200−1400 AD Iñupiat potsherd fragment from Point Barrow was investigated. The described method succeeds in identifying four peptides corresponding to the harbor seal myoglobin (species Phoca vitulina) with a measured mass accuracy better than 1 ppm (MS and MS/MS experiments) including one specific peptide of the cetacean and pinniped species and one specific peptide of the seal species. These results highlight, for the first time, a methodology able to identify proteins from a few milligrams of archeological potsherd buried for years; the obtained results confirm the presence of a seal muscle tissue protein in this Punuk potsherd.

Proteomics and Coast Salish blankets: a tale of shaggy dogs?

by Caroline Solazzo

http://news.sciencemag.org/sciencenow/2011/11/native-american-blankets-made-wi.html

http://www2.macleans.ca/2011/12/16/a-19th-century-canine-mystery-solved/

Identifying animals to species from relict proteins is a powerful new archaeological tool. Here the authors apply the... more Identifying animals to species from relict proteins is a powerful new archaeological tool. Here the authors apply the method to answer questions relating to the Salish of west coast North America.Did they weave their blankets out of dog hair? The proteomic analysis shows that they did, interweaving it with goat, and that the woolly dog was increasingly superseded by sheep in the later nineteenth century.

In silico modification of suberoylanilide hydroxamic acid (SAHA) as potential inhibitor for class II histone deacetylase (HDAC)

by Arli Parikesit

Co-authored with Usman Sumo Friend Tambunan and Bramantya

Background: The cervical cancer is the second most prevalent cancer for the woman in the world. It is caused by the... more Background: The cervical cancer is the second most prevalent cancer for the woman in the world. It is caused by the oncogenic human papilloma virus (HPV). The inhibition activity of histone deacetylase (HDAC) is a potential strategy for cancer therapy. Suberoylanilide hydroxamic acid (SAHA) is widely known as a low toxicity HDAC inhibitor. This research presents in silico SAHA modification by utilizing triazole, in order to obtain a better inhibitor. We conducted docking of the SAHA inhibitor and 12 modified versions to six class II HDAC enzymes, and then
proceeded with drug scanning of each one of them.
Results: The docking results show that the 12 modified inhibitors have much better binding affinity and inhibition potential than SAHA. Based on drug scan analysis, six of the modified inhibitors have robust pharmacological attributes, as revealed by drug likeness, drug score, oral bioavailability, and toxicity levels.
Conclusions: The binding affinity, free energy and drug scan screening of the best inhibitors have shown that 1c
and 2c modified inhibitors are the best ones to inhibit class II HDAC

Integrated Analysis of Genetic, Genomic and Proteomic Data

by Bill White

The rapid expansion of methods for measuring biological data ranging from DNA sequence variations to mRNA expression... more The rapid expansion of methods for measuring biological data ranging from DNA sequence variations to mRNA expression and protein abundance presents the opportunity to utilize multiple types of information jointly in the study of human health and disease. Organisms are complex systems that integrate inputs at myriad levels to arrive at an observable phenotype. Therefore, it is essential that questions concerning the etiology of phenotypes as complex as common human diseases take the systemic nature of biology into account, and integrate the information provided by each data type in a manner analogous to the operation of the body itself. While limited in scope, the initial forays into the joint analysis of multiple data types have yielded interesting results that would not have been reached had only one type of data been considered. These early successes, along with the aforementioned theoretical appeal of data integration, provide impetus for the development of methods for the parallel, high-throughput analysis of multiple data types. The idea that the integrated analysis of multiple data types will improve the identification of biomarkers of clinical endpoints, such as disease susceptibility, is presented as a working hypothesis

Protein expression profile of Gluconacetobacter diazotrophicus PAL5, a sugarcane endophytic plant growth‐promoting bacterium

by Marcelo Bertalan

Proteome turnover in the green alga Ostreococcus tauri by time course 15N metabolic labeling mass spectrometry

by Sarah Martin

Journal of Proteome Research
Martin SF, Munagapati VS, Salvo-Chirnside E, Kerr L, Le Bihan, T

Protein synthesis and degradation determine the cellular levels of proteins, and their control hence enables organisms... more Protein synthesis and degradation determine the cellular levels of proteins, and their control hence enables organisms to respond to environmental change. Experimentally, these are little known proteome parameters, however recently, SILAC-based mass spectrometry studies have begun to quantify turnover in the proteomes of cell lines, yeast and animals. Here, we present a proteome-scale method to quantify turnover and calculate synthesis and degradation rate constants of individual proteins in autotrophic organisms such as algae and plants. The workflow is based on the automated analysis of partial stable isotope incorporation with ¹⁵N. We applied it in a study of the unicellular pico-alga <i>Ostreococcus tauri</i> and observed high relative turnover in chloroplast-encoded ATPases (0.42%-0.58% h^-1), core photosystem II proteins (0.34%-0.51% h^-1) and RbcL (0.47% h^-1), while nuclear-encoded RbcS2 is more stable (0.23% h^-1). Mitochondrial targeted ATPases (0.14%-0.16% h^-1), photosystem antennae (0.09%-0.14% h^-1) and histones (0.07%-0.1% h^-1) were comparatively stable. The calculation of degradation and synthesis rate constants k_deg and k_syn confirms RbcL as the bulk contributor to overall protein turnover. This study performed over 144h of incorporation reveals dynamics of protein complex subunits as well as isoforms targeted to different organelles.

Proteomic analysis of mitochondria in APOE transgenic mice and in response to an ischemic challenge

by Sarah Martin

Journal of Cerebral Blood Flow & Metabolism
James R, Searcy JL, Le Bihan T, Martin SF, Gliddon CM, Povey J, Deighton RF, Kerr LE, McCulloch J, Horsburgh K.

Apolipoprotein E (APOE)-ɛ4 is associated with a deleterious outcome after ischemic brain injury, which may involve... more Apolipoprotein E (APOE)-ɛ4 is associated with a deleterious outcome after ischemic brain injury, which may involve abnormal regulation of mitochondrial function. We have assessed the mitochondrial proteomic response of APOE-ɛ3 and APOE-ɛ4 transgenic mice to transient global ischemic injury in the hippocampus. A genotype-dependent increase in ApoE levels in mitochondria was observed after ischemia, with APOE-ɛ4 mice showing significantly greater increases than APOE-ɛ3 mice. Quantitative analysis of the mitochondria-enriched fractions was performed using liquid-chromatography mass spectrometry coupled to label-free analysis. Of the 1,067 identified proteins, 274 were mitochondria associated. Mitochondrial protein expression was significantly different between genotypes under basal conditions as well as in response to global ischemia. A total of 12 mitochondrial proteins (including respiratory chain proteins NDUFA11, NDUFS3, NDUF5B, ATP5J, as well as ETFA, CYB5B, ATP6V1A, HSPA1B, OXR1, GLUL, IARS2, and PHYHIPL) were significantly altered with respect to genotype, global ischemia, or their interaction (P<0.01). A compelling interactome, created using proteins found to be significantly modulated by global ischemia (P<0.05), involved proteins that regulate energy production and oxidative stress. Thus, APOE genotype has a differential effect on the mitochondrial protein expression in the absence and presence of an injury, which may underlie the differing genotype susc

Evolution and Quantitative Comparison of Genome-Wide Protein Domain Distributions

by Arli Parikesit

Co-authored with Peter F Stadler and Sonja J Prohaska

The metabolic and regulatory capabilities of an organism are implicit in its protein content. This is often hard to... more The metabolic and regulatory capabilities of an organism are implicit in its protein content. This is often hard to estimate, however, due to ascertainment biases inherent in the available genome annotations. Its complement of recognizable functional protein domains and their combinations convey essentially the same information and at the same time are much more readily accessible, although protein domain models trained for one phylogenetic group frequently fail on distantly related sequences. Pooling related domain models based on their GO-annotation in combination with de novo gene prediction methods provides estimates that seem to be less affected by phylogenetic biases. We show here for 18 diverse representatives from all eukaryotic kingdoms that a pooled analysis of the tendencies for co-occurrence or avoidance of protein domains is indeed feasible. This type of analysis can reveal general large-scale patterns in the domain co-occurrence and helps to identify lineage-specific variations in the evolution of protein domains. Somewhat surprisingly, we do not find strong ubiquitous patterns governing the evolutionary behavior of specific functional classes. Instead, there are strong variations between the major groups of Eukaryotes, pointing at systematic differences in their evolutionary constraints.