Gamers succeed where scientists fail

"Gamers have solved the structure of a retrovirus enzyme whose configuration had stumped scientists for more than a decade. The gamers achieved their discovery by playing Foldit, an online game that allows players to collaborate and compete in predicting the structure of protein molecules."

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A lack of structure facilitates protein synthesis

Texts without spaces are not very legible, as they make it very difficult for the reader to identify where a word begins and where it ends. When genetic information in our cells is read and translated into proteins, the enzymes responsible for this task face a similar challenge. They must find the correct starting point for protein synthesis. Therefore, in organisms with no real nucleus, a point exists shortly before the start codon, to which the enzymes can bind particularly well. This helps them find the starting point itself. However, genes that do not have this sequence are also reliably translated into proteins. Scientists from the Max Planck Institute of Molecular Plant Physiology in Potsdam have discovered that the structure of the messenger RNA probably plays a crucial role in this process.

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Roche´s xCELLigence RTCA HT System: Fully-automated Measurement of Therapeutic Targets` Cellular Activity

"Label-free technologies have entered the stage of cellular drug discovery and high-throughput screening (HTS). For the measurement of G protein-coupled receptor (GPCR) activation electrical impedance represents an excellent universal readout technology, since different signaling pathways can be measured in one assay format using recombinant as well as primary cells. The recently developed xCELLigence RTCA HT Instrument from Roche Applied Science now allows to perform fully-automated impedance screens for GPCRs and other targets in the 384-well high-throughput format.
In a recent case study, Urs Lüthi and John Gatfield from Actelion Pharmaceuticals Ltd., Allschwil, Switzerland, integrated 2 RTCA HT (real-time cell analyzer for high-throughput) Instruments on an automated high-throughput screening platform from Agilent Technologies (Santa Clara, US). 263 antagonist hits of the orexin type 1 (Ox1) GPCR that had been identified in a classical calcium flux (FLIPR) HTS were screened for Ox1 inhibition in fully-automated RTCA HT assays. The overall performance, the quality of E-Plates 384 and intra- and inter-assay reproducibility were evaluated. 65% of the 263 antagonist hits were confirmed to be Ox1 receptor antagonists after impedance measurements. According to the researchers, the RTCA HT Instrument could be readily integrated into automated workflows and delivered a highly reproducible data set, making the RTCA HT Instrument a powerful screening technology. 

Compared to standard readout technologies one of the major advantages of label-free technologies is that cellular processes are measured in real-time kinetics in a non-invasive manner. The xCELLigence System uses gold electrodes at the bottom surface of microplate wells as sensors to which an alternating current is applied. Cells that are grown as adherent monolayers on top of such electrodes influence the alternating current at the electrodes by changing the electrical resistance (impedance). The degree of this change is primarily determined by the number of cells, strength of the cell-cell interactions, interactions of the cells with the microelectrodes and by the overall morphology of the cells."

MSE from Waters - the ultimate technology for reproducible profiling

"Waters mass spectrometers provide a method of data acquisition - known as MS^E - that records exact mass precursor and fragment ion information from every detectable component in a sample. This method rapidly alternates between two functions: the first acquiring low-energy exact mass precursor ion spectra, the second acquiring elevated-energy exact mass fragment ion spectra. Every mass is measured, and spectra for each component aligned in retention time. This patented method records data without discrimination or pre-selection so your samples are completely catalogued in a single analysis.



When compared to Data Directed Analysis (DDA), MS^E maximizes instrument duty cycle by ensuring that exact mass precursor and fragment ion information data are obtained for the entire peak complement of a chromatogram, making it ideal for fast analysis and narrow, rapidly eluting peaks. DDA results in both a loss of data in the MS mode when MS/MS data are being acquired, and poor duty cycle. MS^E data is collected fast enough to accurately define the LC peaks for every detectable component.



MS^E is faster than traditional MS followed by MS/MS analysis, and provides data that is not readily obtained by DDA, as both MS and MS/MS data for all detectable components in the chromatogram are generated. MS^E can generate both precursor and product ions in a single analytical run thereby eliminating the need to rerun the samples to obtain further MS/MS spectra. To see MS^E in action and hear what scientists have to say about it, visit www.waters.com/MS^E."

Thermo Fisher Scientific Introduces Peptide Calibration Reagent to Optimize Liquid Chromatography Performance

"Thermo Fisher Scientific, the world leader in serving science, today announced the availability of the Thermo Scientific Pierce Peptide Retention Time Calibration Mixture for the prediction of peptide retention times on reversed-phase high-performance liquid chromatography (HPLC) columns. 
The convenient, ready-to-use Pierce® Peptide Retention Time Calibration Mixture contains 15 synthetic, heavy peptides mixed at an equimolar ratio to elute across the chromatographic gradient. It can be used with Thermo Scientific Pinpoint Software to predict peptide retention time from sequence alone, using hydrophobicity factors, or to predict peptide retention time between instrument platforms. 

The Pierce Peptide Retention Time Calibration Mixture streamlines the transition from qualitative protein discovery results to the development of targeted mass spectrometry (MS) assays on Thermo Scientific Triple Quadrupole, Orbitrap and Exactive Instruments and all other mass spectrometers. It also saves time in peptide purification by increasing the prediction efficiency of peptide retention profiles. The mixture is useful in evaluating different reversed-phase column and gradient options, monitoring for autosampler and HPLC column performance characteristics and normalizing results between experiments and over time."

Published Study Validates New Protein Enrichment Approach For Low-Abundance Biomarker Detection

Hercules, CA — April 20, 2011 — University of Minnesota researchers found that Bio-Rad Laboratories' ProteoMiner protein enrichment kit enhanced identification of changes to low-abundance proteins and detection of post-translationally modified (PTM) proteins in human saliva. These findings offer promise for improving differential proteomic analyses and biomarker studies aimed at identifying disease-specific proteins and their PTM variants in various types of biological samples and fluids. The study was published in the Dec. 13, 2010, issue of the Journal of Proteome Research.

ven when highly sensitive mass spectrometers are used to analyze complex biological samples and bodily fluids, high-abundance proteins obscure the detection of lower-abundance proteins and their post-translational modifications," said Sri Bandhakavi, who led the study at the University of Minnesota in 2010. (Bandhakavi is now a senior scientist at Bio-Rad.) "These lower-abundance proteins and PTMs are often of most interest to researchers, given their association with specific disease or physiological states."

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Network-Based Pipeline for Analyzing MS Data: An Application toward Liver Cancer

Current limitations in proteome analysis by high-throughput mass spectrometry (MS) approaches have sometimes led to incomplete (or inconclusive) data sets being published or unpublished. In this work, we used an iTRAQ reference data on hepatocellular carcinoma (HCC) to design a two-stage functional analysis pipeline to widen and improve the proteome coverage and, subsequently, to unveil the molecular changes that occur during HCC progression in human tumorous tissue. The first involved functional cluster analysis by incorporating an expansion step on a cleaned integrated network. The second used an in-house developed pathway database where recovery of shared neighbors was followed by pathway enrichment analysis. In the original MS data set, over 500 proteins were detected from the tumors of 12 male patients, but in this paper we reported an additional 1000 proteins after application of our bioinformatics pipeline. Through an integrative effort of network cleaning, community finding methods, and network analysis, we also uncovered several biologically interesting clusters implicated in HCC. We established that HCC transition from a moderate to poor stage involved densely connected clusters that comprised of PCNA, XRCC5, XRCC6, PARP1, PRKDC, and WRN. From our pathway enrichment analyses, it appeared that the HCC moderate stage, unlike the poor stage, is enriched in proteins involved in immune responses, thus suggesting the acquisition of immuno-evasion. Our strategy illustrates how an original oncoproteome could be expanded to one of a larger dynamic range where current technology limitations prevent/limit comprehensive proteome characterization.

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Strong Investments to Boost Global Bioinformatics Market

According to our latest report entitled “Global Bioinformatics Market Outlook”, the global bioinformatics industry has been witnessing a remarkable growth rate pattern for the past few years. Factors, such as increasing R&D investments by companies and regulatory support boosted the market revenue to reach around US$ 2.6 Billion in 2009. As the bioinformatics market is at the nascent stage, its full potential is yet to be exploited. Moreover, there are significant advances in technologies that will boost the bioinformatics industry in future. It is expected that the market will grow at a CAGR of nearly 26% during 2011-2013 to reach US$ 6.2 Billion.

Our report has found that the escalating importance of personalized medicines has significantly augmented the growth level in proteomics market. Rising awareness of protein research and its study has opened the market for different proteomic-related equipments, technologies, and services. It is estimated that by 2013, the proteomics market will be worth around US$ 17 Billion and it will act as an important contributor in the growth of bioinformatics.

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Super-SILAC Technology for Quantitative Proteomics in Neoplasms

A group of investigators under Dr. Matthias Mann at Max Planck Institute of Biochemistry in Martinsried, Germany is working on an interesting quantitative proteomics technology that might offer a new way to analyze cell proteins in a range of disorders, such as cancer and autoimmune diseases. Called super-SILAC (stable-isotope labeling by amino acids in cell culture), the method generates thousands of isotopically labeled peptides in unique amounts to serve as "internal standards for mass spectrometry-based analysis."

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Research scientists develop powerful new methodology for stabilizing proteins

A team of scientists at The Scripps Research Institute has discovered a new way to stabilize proteins - the workhorse biological macromolecules found in all organisms. Proteins serve as the functional basis of many types of biologic drugs used to treat everything from arthritis, anemia, and diabetes to cancer.

As described in the February 4, 2011 edition of the journal Science, when the team attached a specific oligomeric array of sugars called a "glycan" to proteins having a defined structure, the proteins were up to 200 times more stable in the test tube. In the body, this stability may translate into longer half-lives for therapies, possibly lowering the overall cost of treatment for certain protein-based drugs and requiring patients to have fewer injections during a course of treatment.

The work may have major implications for the drug industry because there are a large number of protein-based drugs on the market, more in clinical trials, and many more under development worldwide. Nearly all of these protein-based drugs have glycans attached to them and are therefore called "glycoproteins". Glycoprotein-based drugs can be quite expensive to produce and usually need to be administered intravenously.

One of the challenges in producing these drugs has been increasing their stability, which generally extends their half-life in the bloodstream - issues that the new discovery appears to address directly.

"We've now provided engineering guidelines for glycoprotein stability," said Scripps Research Professor Jeffery W. Kelly, who is chair of the Department of Molecular and Experimental Medicine, Lita Annenberg Hazen Professor of Chemistry, and member of The Skaggs Institute for Chemical Biology at Scripps Research. Kelly led the study with Scripps Research Associate Professor Evan Powers and Staff Scientist Sarah R. Hanson, in collaboration with Research Associates Elizabeth K. Culyba, Joshua Price, and colleagues.

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Canadian Human Proteome Project Workshop

Canadian Human Proteome Project Workshop Part 2, MaRS District Discovery, TorontoFebruary 22, 2011
MaRS District Discovery
Toronto, Ontario

The Canadian National Proteomics Network is promoting a Canadian Human Proteomics Project (CHPP), which will leverage Canada’s existing strengths in proteomics, health research and technology. A position paper is currently being developed based on the outcomes of the first CHPP Workshop held on January 16-17, 2011 in conjunction with the Annual Proteomics Conference in Cell Biology in Barbados. The CHPP Working Group will present the first draft of this position paper and invite feedback and further development of the project from the research community, clinicians and industry partners. As members of the CNPN and CNPN Board of Directors, the goal of the organizers of the Workshop is to present a final version of the position paper to Canadian and provincial funding agencies in the Spring of 2011 with the intent of stimulating interest for a nationwide funding competition to support a CHPP.

We invite you to participate in the development of a CHPP during our full-day Workshop on February 22. The Workshop will consist of presentations from leaders around the world participating in the Human Proteome Project, discussion of the goals and potential projects for CHPP, and key strategies for engaging national funding agencies and industry partners. 

Highly Sensitive & Specific Chromatin Immunoprecipitation (ChIP) Assay Kit from Porvair Filtration Group

orvair Filtration Group has developed a new technology that has particular relevance to the rapidly expanding Epigenetics market and will be unveiling it at the Epigenetics World Congress in Boston in April 2011. Using a new approach, based on a rigid porous polymer matrix rather than the traditional sepharose or magnetic beads, Porvair has developed a novel Chromatin ImmunoPrecipitation (ChIP) assay kit called Chromatrap™.

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Stable isotope shifted matrices enable the use of low mass ion precursor scanning for targeted metabolite identification

We describe a method to identify metabolites of exogenous proteins that eliminates endogenous background by using stable isotope labeled matrices. This technique allows selective screening of the intact therapeutic molecule and all metabolites using a modified precursor ion scan that monitors low molecular weight fragment ions produced during MS/MS. This distinct set of low mass ions differs between isotopically labeled and natural isotope containing species allowing excellent discrimination between endogenous compounds and target analytes during the precursor scanning experiments. All compounds containing amino acids that consist of naturally abundant isotopes can be selected using this scanning technique for further analysis, including metabolites of the parent molecule. The sensitivity and selectivity of this technique is discussed with specific examples of insulin derived peptides being screened from a complex matrix using a range of different validated target ions.


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peptide retention time

Is peptide RT charge specific?
"Not in the sense that ESI-charge is influencing the retention time but
in the sense that longer peptide tend to elute later in the
chromatogram and also are more likely to have higher charge states,
there is a slight correlation between charge state and retention time.
however it would work as prediction tool if you consider predicting
the charge state of a peptide, which is actually not that difficult to
do." by Hannes.

O18 labeling

"If digestion of proteins by trypsin is performed in a solution that contains 50% O16 water and 50% O18 water, then for most peptides there should be approximately a 1 part unlabeled : 2 parts singly labeled : 1 part doubly labeled ratio.

To understand this, one must think about the process of digestion itself. Trypsin cleaves proteins through hydrolysis. The newly-created C-terminus carboxyl acquires one of its oxygen atoms from the water. So, at this point, half of the C-termini will be singly labeled, and half will be unlabeled. (I'm ignoring the low probability of O18 atoms already being present at the site of cleavage).

However, in most cases, trypsin will continue to interact with the C-termini, swapping out C-terminal oxygens to create new water molecules while at the same time swapping in oxygens from existing water molecules to replace them. After this process has proceeded for some time, the chance of a given C-terminal oxygen atom being O16 approaches the level of its presence in the water: i.e. 50%. The same is true for its chance of being an O18 atom.

So, a simple binomial distribution applies. Since the C-terminal carboxyl has two oxygen atoms:

Chance of both being unlabeled = 0.5 times 0.5 = 0.25.
Chance of both being labeled = 0.5 times 0.5 = 0.25
Chance of one being labeled and the other being unlabeled = the remainder of the probability = 1.0 - 0.25 - 0.25 = 0.50.

So, one quarter of the peptides are unlabeled, one half singly-labeled, and one quarter doubly-labeled. A 1:2:1 ratio should be evident.

That being said, different peptides show different rates of back exchange, with a few being totally resistant to it. So while the majority of peptides should display a 1:2:1 ratio pattern, some exceptions might be noted."

From internal communications.

top-down vs. bottom-up protoemics

Bottom-Up Proteomics

In bottom-up proteomics, the analytes introduced into the mass spectrometer are peptides generated by enzymatic cleavage of one or many proteins. The proteins can first be separated by GE or chromatography, in which case the sample will contain only one or a few proteins. Alternatively, a complex protein mixture initially can be digested to the peptide level, then separated by on-line chromatography coupled to electrospray mass spectrometry (ESI–MS). In the latter case, the digest can contain thousands to hundreds of thousands of peptides, and require separation in two or more chromatographic dimensions before MS analysis. The identity of the original protein is determined by comparison of the peptide mass spectra with theoretical peptide masses calculated from a proteomic or genomic database. There are two approaches for protein identification using the bottom-up approach, peptide mass fingerprinting and tandem MS (MS–MS).



Top-Down Proteomics

In top-down proteomics, intact protein molecular ions generated by ESI/MALDI are introduced into the mass analyzer and are subjected to gas-phase fragmentation. An obstacle to this approach is the determination of product ion masses from multiply charged product ions (1). These can vary in charge state up to that of the multiply charged protein precursor ion. This can introduce ambiguity in the interpretation of top-down MS-MS spectra. Two approaches have been used to circumvent this limitation. The first is charge state manipulation through gas phase ion–ion interactions, and the second is the use of instruments with high mass measurement accuracy (MMA). It provides an approach for large scale characterization of proteins, both types of FTMS instruments, ICR and Orbitrap have been used for this methodology. The molecular mass of the inact precursor protein along with fragment ions from MS/MS experiments enables high confidence mapping to database protein entries as well as PTM detection.


Advantages and limitations of bottom-up strategies: Bottom-up proteomics is the most mature and most widely used approach for protein identification and characterization. Reversed-phase HPLC provides high-resolution separations of peptide digests with solvents that are compatible with ESI. On-line nano-scale reversed-phase LC–ESI–MS–MS can be fully automated and is almost universally used for bottom-up proteomics. Commercial instruments with control software and bioinformatics tools optimized for bottom-up applications are available from several vendors. The bottom-up strategy using on-line multidimensional capillary HPLC–MS-MS has been most successful in the identification of proteins in digests derived from very complex mixtures such as cell lysates (6). Moreover, quantitative techniques have been developed using affinity tags and stable isotope labels for determination of up- and down-regulated proteins in expression proteomics (7).
There are several fundamental and practical limitations to the bottom-up strategy.
Most importantly, only a fraction of the total peptide population of a given protein is identified. Therefore, information on only a portion of the protein sequence is obtained. It is clear from genomic studies that each open reading frame can give rise to many protein isoforms, which can originate from alternative splicing products and varying types and locations of posttranslational modifications (PTMs). PTMs such as phosphorylation and glycosylation are known to be important in the regulation of protein function and cell metabolism. A consequence of the limited sequence coverage in bottom-up proteomics is loss of much information about PTMs. Moreover, PTMs are often labile in the CID process and require techniques such as neutral loss scanning to detect them.
Practical limitations are encountered when bottom-up methods are used for protein identification from very complex peptide mixtures. On-line multidimensional LC–MS-MS analyses using ion-exchange coupled to reversed-phase columns require extended run times of as long as 15 h or more. Although this can be automated, the throughput of multidimensional LC–MS-MS is quite limited. Other problems include the loss of information about low-abundance peptides in mass spectra dominated by high-abundance species. Finally, narrow chromatographic peak widths can compromise acquisition of adequate MS–MS information during elution.


Advantages and limitations of top-down strategies: The two major advantages of the top-down strategy are the potential access to the complete protein sequence and the ability to locate and characterize PTMs. In addition, the time-consuming protein digestion required for bottom-up methods is eliminated.
Top-down proteomics is a relatively young field compared to bottom-up proteomics, and currently suffers from several limitations. First, the very complex spectra generated by multiply charged proteins limits the approach to isolated proteins, or simple protein mixtures at best. Second, the favored instrumentation (FT-ICR, hybrid ion trap FT-ICR or hybrid ion trap–orbitrap) are expensive to purchase and operate. Third, the top-down approach does not work well with intact proteins larger than about 50 kDa. Fourth, the favored dissociation techniques (ECT, ETD) are low-efficiency processes requiring long ion accumulation, activation, and detection times. This limits the ability to couple top-down MS techniques with on-line separations. Fifth, the mechanisms of protein dissociation behavior are less well understood than those of peptide dissociation. If top-down approaches are to be adopted widely, a greater understanding of fragmentation of multiply charged ions is needed (1), including the influence of precursor ion charge state, the role of protein primary, secondary and tertiary structure, and the contribution of PTMs. Finally, bioinformatics tools for top-down proteomics are primitive compared to those for bottom-up proteomics.


Thermo Scientific* ProSightPC, the first stand-alone software for analyzing top-down proteomics data, has been enhanced to add support for middle-down and bottom-up experiments, making it an all-around tool for identification and characterization of both intact proteins and peptides.
ProSightPC* 2.0 software enables high-throughput processing of all accurate-mass MS/MS data, whether from top-down, middle-down or bottom-up experiments including the characterization of proteins with known PTMs. ProSightPC 2.0 software uses multiple search modes to determine the exact protein sequence including modifications and alternative splicing. It is the only proteomics software that allows the user to search their tandem MS data against proteome warehouses containing the known biological complexity present in UniProt.

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A new proof of Darwinian Evolution

Motivation: The article presents results of the listing of the quantity of amino acids, dipeptides and tripeptides for all proteins available in the UNIPROT–TREMBL database and the listing for selected species and enzymes. UNIPROT–TREMBL contains protein sequences associated with computationally generated annotations and large-scale functional characterization. Due to the distinct metabolic pathways of amino acid syntheses and their physicochemical properties, the quantities of subpeptides in proteins vary. We have proved that the distribution of amino acids, dipeptides and tripeptides is statistical which confirms that the evolutionary biodiversity development model is subject to the theory of independent events. It seems interesting that certain short peptide combinations occur relatively rarely or even not at all. First, it confirms the Darwinian theory of evolution and second, it opens up opportunities for designing pharmaceuticals among rarely represented short peptide combinations. Furthermore, an innovative approach to the mass analysis of bioinformatic data is presented.


read the full article.

ProSightPC

ProSightPC 2.0 Software

Thermo Scientific* ProSightPC, the first stand-alone software for analyzing top-down proteomics data, has been enhanced to add support for middle-down and bottom-up experiments, making it an all-around tool for identification and characterization of both intact proteins and peptides.
ProSightPC* 2.0 software enables high-throughput processing of all accurate-mass MS/MS data, whether from top-down, middle-down or bottom-up experiments including the characterization of proteins with known PTMs. ProSightPC 2.0 software uses multiple search modes to determine the exact protein sequence including modifications and alternative splicing. It is the only proteomics software that allows the user to search their tandem MS data against proteome warehouses containing the known biological complexity present in UniProt.

ProSightPC 2.0 software is a complete software package for the identification and characterization of proteins, peptides, and PTMs. It features multiple search modes and can accommodated data generated with several different fragmentation techniques.
Supports top-down, middle-down, and bottom-up experiments
Includes five different search modes, including Accurate Mass, Biomarker, Sequence Tag, Single Protein and Gene Restricted search modes
Processes fragmentation data from ECD, IRMPD or CID

The proprietary ProSight Warehouse includes all known post-translational modifications (PTMs), alternative splicing events and single nucleotide polymorphisms (SNPs)
Import FASTA databases and shotgun annotates these databases with all possible modifications
Includes Sequence Gazer, which allows users to review search results and add, remove, or change modifications to look for better fits

Compatible with:
LTQ FT family of hybrid mass spectrometers
LTQ Orbitrap family of hybrid mass spectrometers
Proteome Discoverer software


A user guide