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Mass spectrometry-based identification of proteins interacting with nucleic acids

Mass spectrometry-based identification of proteins interacting with nucleic acids | proteomics | Scoop.it
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The identification of the regulatory proteins that control DNA transcription as well as RNA stability and translation represents a key step in the comprehension of gene expression regulation. Those proteins can be purified by DNA- or RNA-affinity chromatography, followed by identification by mass spectrometry. Although very simple in the concept, this represents a real technological challenge due to the low abundance of regulatory proteins compared to the highly abundant proteins binding to nucleic acids in a nonsequence-specific manner. Here we review the different strategies that have been set up to reach this purpose, discussing the key parameters that should be considered to increase the chances of success. Typically, two categories of biological questions can be distinguished: the identification of proteins that specifically interact with a precisely defined binding site, mostly addressed by quantitative mass spectrometry, and the identification in a non-comparative manner of the protein complexes recruited by a poorly characterized long regulatory region of nucleic acids. Finally, beside the numerous studies devoted to in vitro-assembled nucleic acid–protein complexes, the scarce data reported on proteomic analyses of in vivo-assembled complexes are described, with a special emphasis on the associated challenges.

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The One Hour Yeast Proteome

The One Hour Yeast Proteome | proteomics | Scoop.it

We describe the comprehensive analysis of the yeast proteome in just over one hour of optimized analysis. We achieve this expedited proteome characterization with improved sample preparation, chromatographic separations, and by using a new Orbitrap hybrid mass spectrometer equipped with a mass filter, a collision cell, a high-field Orbitrap analyzer, and, finally, a dual cell linear ion trap analyzer (Q-OT-qIT, Orbitrap Fusion). This system offers high MS2 acquisition speed of 20 Hz and detects up to 19 peptide sequences within a single second of operation. Over a 1.3 hour chromatographic method, the Q-OT-qIT hybrid collected an average of 13,447 MS1 and 80,460 MS2 scans (per run) to produce 43,400 (average) peptide spectral matches and 34,255 (average) peptides with unique amino acid sequences (1% FDR). On average, each one hour analysis achieved detection of 3,977 proteins (1% FDR). We conclude that further improvements in mass spectrometer scan rate could render comprehensive analysis of the human proteome within a few hours.

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Protein Digestion: An Overview of the Available Techniques and Recent Developments - Journal of Proteome Research (ACS Publications)

Protein Digestion: An Overview of the Available Techniques and Recent Developments - Journal of Proteome Research (ACS Publications) | proteomics | Scoop.it

In this review, an overview is given of the currently available digestion strategies and recent developments in the acceleration of the digestion process. Additionally, tailored approaches for classes of proteins that pose specific challenges are discussed.

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Advances in purification and separation of posttranslationally modified proteins

Advances in purification and separation of posttranslationally modified proteins | proteomics | Scoop.it

Posttranslational modifications (PTMs) of proteins represent fascinating extensions of the dynamic complexity of living cells’ proteomes. The results of enzymatically catalyzed or spontaneous chemical reactions, PTMs form a fourth tier in the gene – transcript – protein cascade, and contribute not only to proteins’ biological functions, but also to challenges in their analysis. There have been tremendous advances in proteomics during the last decade. Identification and mapping of PTMs in proteins have improved dramatically, mainly due to constant increases in the sensitivity, speed, accuracy and resolution of mass spectrometry (MS). However, it is also becoming increasingly evident that simple gel-free shotgun MS profiling is unlikely to suffice for comprehensive detection and characterization of proteins and/or protein modifications present in low amounts. Here, we review current approaches for enriching and separating posttranslationally modified proteins, and their MS-independent detection. First, we discuss general approaches for proteome separation, fractionation and enrichment. We then consider the commonest forms of PTMs (phosphorylation, glycosylation and glycation, lipidation, methylation, acetylation, deamidation, ubiquitination and various redox modifications), and the best available methods for detecting and purifying proteins carrying these PTMs. This article is part of a Special Issue entitled: Protein Modifications.

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There is also a preprint open access version for those who do not have JP subscription: https://docs.google.com/file/d/0B0wA1B6itHpkSVNoUlBDVXBWWk0/edit?usp=sharing

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In silico instrumental response correction improves precision of label-free proteomics and accuracy of proteomics-based predictive models

In silico instrumental response correction improves precision of label-free proteomics and accuracy of proteomics-based predictive models | proteomics | Scoop.it

In the analysis of proteome changes arising during the early stages of a biological process (e.g., disease or drug treatment) or from indirect influence of an important factor, the biological variations of interest are often small (~10%). The corresponding requirements for the precision of proteomics analysis are high, and this often poses a challenge - especially when employing label-free quantification. One of the main contributors to the inaccuracy of label-free proteomics experiments is the variability of the instrumental response during LC-MS/MS runs. Such variability may include fluctuations in: electrospray current, transmission efficiency from the air-vacuum interface to the detector, and detection sensitivity. We have developed an in silico post-processing method of reducing these variations, and have thus significantly improved the precision of label-free proteomics analysis. For abundant blood plasma proteins, a coefficient of variation (CV) of ca. 1% was achieved, which allowed for sex differentiation in pooled samples and 90% accurate differentiation of individual samples by a single LC-MS/MS analysis. This method improves the precision of measurements, and increases the accuracy of predictive models based on the measurements. The post-acquisition nature of the correction technique and its generality promise its widespread application in LC-MS/MS based methods, such as proteomics and metabolomics.

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The impact of biosampling procedures on molecular data interpretation

The impact of biosampling procedures on molecular data interpretation | proteomics | Scoop.it

The separation between biological and technical variation without extensive use of technical replicates is often challenging, particularly in the context of different forms of protein and peptide modifications. Biosampling procedures in the research laboratory are easier to conduct within a shorter time frame and under controlled conditions as compared to clinical sampling with the later often having issues of reproducibility. But is the research laboratory biosampling really less variable? Biosampling introduces within minutes rapid tissue specific changes in the cellular microenvironment; inducing a range of different pathways associated with cell survival. Biosampling involves hypoxia and hypothermia which are circumstances for which there are evolutionary conserved defense strategies in range of species and also are relevant for a range of biomedical conditions. It remains unclear to what extent such adaptive processes are reflected in different biosampling procedures or how important they are for the definition of sample quality. Lately, an increasing number of comparative studies on different biosampling approaches, post-mortem effects and pre-sampling biological state have investigated such immediate early biosampling effects. Commonalities between biosampling effects and a range of ischemia/reperfusion and hypometabolism/anoxia associated biological phenomena indicate that even small variations in post-sampling time intervals are likely to introduce a set of non-random and tissue specific effects of experimental importance (both in vivo and in vitro). This review integrates the information provided by these comparative studies and discusses how an adaptive biological perspective in biosampling procedures may be relevant for sample quality issues.

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Using databases and web resources for glycomics research

Using databases and web resources for glycomics research | proteomics | Scoop.it

Many carbohydrate structure and related databases can be found across the World Wide Web. This review covers the major carbohydrate databases that have potential for utility by glycoscientists and by researchers entering the glycosciences. The first half provides a brief overview of carbohydrate databases and web resources (including a history of carbohydrate databases and carbohydrate notations used in these databases), and the second half provides a guide that can be used as an index to determine which resources provide the data of most interest to the user.

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The Rise of Dinosaur Proteomics? « Promega Connections

The Rise of Dinosaur Proteomics? « Promega Connections | proteomics | Scoop.it
Years ago when I was still working in the lab, I was looking for control RNA options for my experiment in the Ambion catalog when I came across a listing for dinosaur brain RNA and DNA. I had to call their customer service ...
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N-terminal protein processing: A comparative proteogenomic analysis

N-terminal protein processing: A comparative proteogenomic analysis | proteomics | Scoop.it

N-terminal methionine excision (NME) and N-terminal acetylation (NTA) are two of the most common protein post-translational modifications. NME is a universally conserved activity and a highly specific mechanism across all life forms. NTA is very common in eukaryotes but occurs rarely in prokaryotes. By analyzing datasets from yeast, mammals and bacteria (including 112 million spectra from 57 bacterial species), the largest comparative proteogenomics study to date, it is shown that previous assumptions/perceptions about the specificity and purposes of NME are not entirely correct. Although NME, through the universal enzymatic specificity of the methionine aminopeptidases, results in the removal of the initiator Met in proteins when the second residue is Gly, Ala, Ser, Cys, Thr, Pro, or Val, the comparative genomic analyses suggest that this specificity may vary modestly in some organisms. In addition, the functional role of NME may be primarily to expose Ala and Ser rather than all seven of these residues. While any of this group provide ``stabilizing'' N-termini in the N-end rule, and de facto leave the remaining thirteen amino acid types that are classed as ``destabilizing'' (in higher eukaryotes) protected by the initiator Met, the conservation of NME-substrate proteins through evolution suggests that the other five are not crucially important for proteins with these residues in the second position. They are apparently merely inconsequential players (their function is not affected by NME) that become exposed because their side chains are smaller or comparable to those of Ala and Ser. The importance of exposing mainly two amino acids at the N-terminus, i.e. Ala and Ser, is unclear but may be related to NTA or other post-translational modifications. In this regard, these analyses also reveal that NTA is more prevalent in some prokaryotes than previously appreciated.

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File formats commonly used in mass spectrometry proteomics

File formats commonly used in mass spectrometry proteomics | proteomics | Scoop.it

The application of mass spectrometry (MS) to the analysis of proteomes has enabled the high-throughput identification and abundance measurement of hundreds to thousands of proteins per experiment. However, the formidable informatics challenge associated with analyzing MS data has required a wide variety of data file formats to encode the complex data types associated with MS workflows. These formats encompass the encoding of input instruction for instruments, output products of the instruments, and several levels of information and results used by and produced by the informatics analysis tools. A brief overview of the most common file formats in use today is presented along with a discussion of related topics.

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The ubiquitin–proteasome system: central modifier of plant signalling - Sadanandom - 2012 - New Phytologist - Wiley Online Library

The ubiquitin–proteasome system: central modifier of plant signalling - Sadanandom - 2012 - New Phytologist - Wiley Online Library | proteomics | Scoop.it

Ubiquitin is well established as a major modifier of signalling in eukaryotes. However, the extent to which plants rely on ubiquitin for regulating their lifecycle is only recently becoming apparent. This is underlined by the over-representation of genes encoding ubiquitin-metabolizing enzymes in Arabidopsis when compared with other model eukaryotes. The main characteristic of ubiquitination is the conjugation of ubiquitin onto lysine residues of acceptor proteins. In most cases the targeted protein is rapidly degraded by the 26S proteasome, the major proteolysis machinery in eukaryotic cells. The ubiquitin–proteasome system is responsible for removing most abnormal peptides and short-lived cellular regulators, which, in turn, control many processes. This allows cells to respond rapidly to intracellular signals and changing environmental conditions. This review maps out the roles of the components of the ubiquitin–proteasome system with emphasis on areas where future research is urgently needed. We provide a flavour of the diverse aspects of plant lifecycle where the ubiquitin–proteasome system is implicated. We aim to highlight common themes using key examples that reiterate the importance of the ubiquitin–proteasome system to plants. The future challenge in plant biology is to define the targets for ubiquitination, their interactors and their molecular function within the regulatory context.

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bioDBnet - Biological Database Network

bioDBnet - Biological Database Network | proteomics | Scoop.it
bioDBnet is a comprehensive resource of most of the biological databases available from different sites like NCBI, Uniprot, EMBL, Ensembl, Affymetrix.

 

Number of converters - e.g. database:database, including conversion of protein IDs to EC numbers.

 

Summary: bioDBnet is an online web resource that provides interconnected access to many types of biological databases. It has integrated many of the most commonly used biological databases and in its current state has 153 database identifiers (nodes) covering

all aspects of biology including genes, proteins, pathways and other biological concepts. bioDBnet offers various ways to work with these databases including conversions, extensive database reports, custom navigation and has various tools to enhance the quality of
the results. Importantly, the access to bioDBnet is updated regularly, providing access to the most recent releases of each individual database.

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Discovery of O-GlcNAc-6-phosphate-modified proteins by re-analyzing large-scale phosphoproteomics data

Discovery of O-GlcNAc-6-phosphate-modified proteins by re-analyzing large-scale phosphoproteomics data | proteomics | Scoop.it

Phosphorylated O-GlcNAc is a novel posttranslational modification that has so far only been found on the neuronal protein AP180 from the rat (Graham et al., J. Proteome Res. 2011, 10, 2725-33). Upon collision induced dissociation, the modification generates a highly mass deficient fragment ion (m/z 284.0530) that can be used as a reporter for the identification of phosphorylated O-GlcNAc. Using a publically available mouse brain phosphoproteome data set, we employed our recently developed Oscore software to re-evaluate high resolution/high accuracy tandem mass spectra and discovered the modification on 23 peptides corresponding to 11 mouse proteins. The systematic analysis of 220 candidate phosphoGlcNAc tandem mass spectra as well as a synthetic standard enabled the dissection of the major phosphoGlcNAc fragmentation pathways, suggesting that the modification is O-GlcNAc-6-phosphate. We find that the classical O-GlcNAc modification often exists on the same peptides indicating that O-GlcNAc-6-phosphate may biosynthetically arise in two steps involving the O-GlcNAc transferase and a currently unknown kinase. Many of the identified proteins are involved in synaptic transmission and for Ca2+/calmodulin kinase IV, the O-GlcNAc-6-phosphate modification was found in the vicinity of two autophosphorylation sites required for full activation of the kinase suggesting a potential regulatory role for O-GlcNAc-6-phosphate. By re-analysing mass spectrometric data from human embryonic and induced pluripotent stem cells, our study also identified Zinc finger protein 462 (ZNF462) as the first human O-GlcNAc-6-phosphate modified protein. Collectively, the data suggests that O-GlcNAc-6-phosphate is a general post-translation modification of mammalian proteins with a variety of possible cellular functions.

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Plant Proteomics - Methods and Protocols

Plant Proteomics - Methods and Protocols | proteomics | Scoop.it

Plant Proteomics: Methods and Protocols, Second Edition presents recent advances made in the field of proteomics and their application to plant biology and translational research. In recent years, improvements in techniques and protocols for high-throughput proteomics have been made at all workflow stages, from wet (sampling, tissue and cell fractionation, protein extraction, depletion, purification, separation, MS analysis, quantification) to dry lab (experimental design, algorithms for protein identification, bioinformatics tools for data analysis, databases, and repositories). Divided into nine convenient sections, chapters cover topics such as applications of gel-free, label- or label-free, imaging and targeted approaches to experimental model systems, crops and orphan species, as well as the study and analysis of PTMs, protein interactions, and specific families of proteins, and finally proteomics in translational research. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls.

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An Automated Proteogenomic Method Utilizes Mass Spectrometry to Reveal Novel Genes in Zea mays

An Automated Proteogenomic Method Utilizes Mass Spectrometry to Reveal Novel Genes in Zea mays | proteomics | Scoop.it

The pace of genome sequencing is accelerating, revealing the genetic background of a growing number of organisms. However, assigning function to each nucleotide in a completed genome remains the rate-limiting step. New technologies in transcriptomics and proteomics have influenced the emergence of proteogenomics, a field at the confluence of genomics, transcriptomics, and proteomics. First generation proteogenomic toolkits employ peptide mass spectrometry to identify novel protein coding. These efforts rely heavily on existing algorithms designed for standard proteome analysis and fail to address the challenges specific to genome annotation. In this work we extend first generation proteogenomic tools to achieve greater accuracy and enable the analysis of large, complex genomes. We apply our pipeline to Zea mays, which has a genome comparable in size to human. Our pipeline begins with the comparison of mass spectra to a putative translation of the genome. Our translation includes the six-frame translation as well as a splice graph for capturing protein splice variants. We distribute the identification of mass spectra across 45 compute nodes for increased efficiency and employ a database-independent scoring method to improve sensitivity. We select novel peptides, those that match to a region of the genome that was not previously known to be protein coding, for grouping into events. Each of our eight event types describes a refinement needed to the genome annotation. We present a novel, Bayesian framework for evaluating the accuracy of each event. Our calculated event probability, or eventProb, considers the number of supporting peptides and spectra, and the quality of each supporting peptide-spectrum match. More than 80% of the maize genome is comprised of repetitive elements. To address this, our eventProb handles uniquely located peptides and shared location peptides separately. Our pipeline predicts 165 novel protein-coding genes and proposes updated models for 741 additional genes.

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Quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis

Quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis | proteomics | Scoop.it

The majority of mass spectrometry-based protein quantification studies uses peptide-centric analytical methods and thus strongly relies on efficient and unbiased protein digestion protocols for sample preparation. We present a novel objective approach to assess protein digestion efficiency using a combination of qualitative and quantitative LC-MS/MS methods and statistical data analysis. In contrast to previous studies we employed both standard qualitative as well as data-independent quantitative workflows to systematically assess trypsin digestion efficiency and bias using mitochondrial protein fractions. We evaluated nine trypsin-based digestion protocols, based on standard in-solution or on spin filter-aided digestion, including new optimized protocols. We investigated various reagents for protein solubilization and denaturation (dodecyl sulfate, deoxycholate, urea), several trypsin digestion conditions (buffer, RapiGest, deoxycholate, urea), and two methods for removal of detergents prior to analysis of peptides (acid precipitation or phase separation with ethyl acetate). Our data-independent quantitative LC-MS/MS workflow quantified over 3700 distinct peptides with 96% completeness between all protocols and replicates, with an average 40% protein sequence coverage and an average of 11 peptides identified per protein. Systematic quantitative and statistical analysis of physicochemical parameters demonstrated that deoxycholate-assisted in-solution digestion combined with phase transfer allows for efficient, unbiased generation and recovery of peptides from all protein classes, including membrane proteins. This deoxycholate-assisted protocol was also optimal for spin filter-aided digestions as compared to existing methods.

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Combining Results of Multiple Search Engines in Proteomics

Combining Results of Multiple Search Engines in Proteomics | proteomics | Scoop.it

A crucial component of the analysis of shotgun proteomics datasets is the search engine, an algorithm that attempts to identify the peptide sequence from the parent molecular ion that produced each fragment ion spectrum in the dataset. There are many different search engines, both commercial and open source, each employing a somewhat different technique for spectrum identification. Using these search engines, a resultant set of high scoring peptide-spectrum matches for a defined set of input spectra differs markedly among the various search engine results; individual engines each provide unique correct identifications amongst a core set of correlative identifications. This has led to the approach of combining the results from multiple search engines to achieve improved analysis of each dataset. Here we review the techniques and available software for combining the results of multiple search engines and briefly compare the relative performance of these techniques.

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Proteoform: a single term describing protein complexity

Proteoform: a single term describing protein complexity | proteomics | Scoop.it

Smith, Kellner et al. propose that "the term 'proteoform' be used to designate all of the different molecular forms in which the protein product of a single gene can be found, including changes due to genetic variations, alternatively spliced RNA transcripts and post-translational modifications"

 

 

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Why do cellular proteins linked to K63-polyubiquitin chains not associate with proteasomes?

Why do cellular proteins linked to K63-polyubiquitin chains not associate with proteasomes? | proteomics | Scoop.it

Although cellular proteins conjugated to K48-linked Ub chains are targeted to proteasomes, proteins conjugated to K63-ubiquitin chains are directed to lysosomes. However, pure 26S proteasomes bind and degrade K48- and K63-ubiquitinated substrates similarly. Therefore, we investigated why K63-ubiquitinated proteins are not degraded by proteasomes. We show that mammalian cells contain soluble factors that selectively bind to K63 chains and inhibit or prevent their association with proteasomes. Using ubiquitinated proteins as affinity ligands, we found that the main cellular proteins that associate selectively with K63 chains and block their binding to proteasomes are ESCRT0 (Endosomal Sorting Complex Required for Transport) and its components, STAM and Hrs. In vivo, knockdown of ESCRT0 confirmed that it is required to block binding of K63-ubiquitinated molecules to the proteasome. In addition, the Rad23 proteins, especially hHR23B, were found to bind specifically to K48-ubiquitinated proteins and to stimulate proteasome binding. The specificities of these proteins for K48- or K63-ubiquitin chains determine whether a ubiquitinated protein is targeted for proteasomal degradation or delivered instead to the endosomal-lysosomal pathway.

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Global Subcellular Characterisation of Protein Degradation using Quantitative Proteomics

Global Subcellular Characterisation of Protein Degradation using Quantitative Proteomics | proteomics | Scoop.it
Protein degradation provides an important regulatory mechanism used to control cell cycle progression and many other cellular pathways. To comprehensively analyse the spatial control of protein degradation in U2OS osteosarcoma cells, we have combined drug treatment and SILAC-based quantitative mass spectrometry with subcellular and protein fractionation. The resulting dataset analysed more than 74,000 peptides, corresponding to ~5,000 proteins, from nuclear, cytosolic, membrane and cytoskeletal compartments. These data identified rapidly degraded proteasome targets and highlighted a feedback mechanism resulting in translation inhibition, induced by blocking the proteasome. We show this is mediated by activation of the unfolded protein response. We observed compartment-specific differences in protein degradation, including proteins that would not have been characterised as rapidly degraded through analysis of whole cell lysates. Bioinformatics analysis of the entire dataset is presented in the Encyclopedia of Proteome Dynamics, a web-based resource, with proteins annotated for stability and subcellular distribution.
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Label-free proteomics – the protease matters

Label-free proteomics – the protease matters | proteomics | Scoop.it
Trypsin is the most commonly used protease for mass spectrometry–based proteomics experiments, because of its well defined specificity (it results in peptides with either lysine or arginine at the C-termini).

With an increasing focus on answering the question “How many copies of this protein are present per cell?”, it becomes important to make sure that the protease digestion does not result in more- or fewer-than-expected peptides for any given protein.

Is trypsin always the best protease for such proteomics experiments?

It turns out that it would be a good idea to try other proteases as well.

In a letter to Nature Methods, Peng et al. describe results from an experiment where they treated aliquots from a yeast lysate with different proteases (trypsin, Lys-C, Lys-N and chymotrypsin). They then performed strong cation exchange chromatography followed by LC–MS/MS and generated proteomics data sets based on both spectral counts and ion volume. While there was a good correlation between the results obtained from technical replicates, correlation between samples obtained using different proteases was less good, an effect which was especially pronounced with chymotrypsin, which has a different cleavage specificity.

The authors found that while amount of some proteins was similar no matter what protease was used (e.g. those shown in (i) below), there were others where using trypsin seemingly over-estimated the copy number (ii) OR underestimated the copy number (iii), and there were proteins which would not have been seen if only one of the proteases had been used (iv).

(the Supplementary information provides a lot of information regarding the methods used, and includes a more comprehensive set of results).

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Targeted Proteomic Quantification on Quadrupole-Orbitrap Mass Spectrometer

Targeted Proteomic Quantification on Quadrupole-Orbitrap Mass Spectrometer | proteomics | Scoop.it

There is an immediate need for improved methods to systematically and precisely quantify large sets of peptides in complex biological samples. While to date protein quantification in biological samples is routinely performed on triple quadrupole instruments operated in selected reaction monitoring mode (SRM), two major challenges remain. Firstly, the number of peptides to be included in one survey experiment needs to be increased to routinely reach several hundreds, and secondly, the degree of selectivity should be improved to reliably discriminate the targeted analytes from background interferences. High resolution and accurate mass (HR/AM) analysis on the recently developed Q-Exactive mass spectrometer can potentially address these issues. This instrument presents a unique configuration: it is constituted of the orbitrap mass analyzer equipped with a quadrupole mass filter as front-end for precursor ion mass selection. This configuration enables new quantitative methods based on HR/AM measurements, including targeted analysis in MS mode (single ion monitoring, SIM) and in MS/MS mode (parallel reaction monitoring, PRM). While the ability of the quadrupole to select a restricted m/z range allows overcoming the dynamic range limitations associated with trapping devices, the MS/MS mode provides an additional stage of selectivity. When applied to targeted protein quantification in urine samples and benchmarked with the reference SRM technique, the quadrupole-orbitrap instrument exhibits similar or better performances in terms of selectivity, dynamic range, and sensitivity. These high performances are further enhanced by leveraging the multiplexing capability of the instrument to design novel acquisition methods and apply them to large targeted proteomic studies for the first time, as demonstrated on 770 tryptic yeast peptides analyzed in one 60 minute experiment. The increased quality of quadrupole-orbitrap data has the potential to improve existing protein quantification methods in complex samples and to cope with the pressing demand of systems biology or biomarker evaluation studies.

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J-Express Pro - Gene expression analysis software

J-Express Pro - Gene expression analysis software | proteomics | Scoop.it

Genomics software suitable for proteomics analyses. Registration (e-mail + address) unlocks statistics, including PCA analysis and self-organizing maps. Can't replace advanced instruments like Origin, but it is useful for quick overview and seems also more user-friendly.

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Expert System for Computer Assisted Annotation of MS/MS Spectra

Expert System for Computer Assisted Annotation of MS/MS Spectra | proteomics | Scoop.it

An important step in mass spectrometry (MS)-based proteomics is the identification of peptides by their fragment spectra. Regardless of the identification score achieved, almost all MS/MS spectra contain remaining peaks that are not assigned by the search engine. These peaks may be explainable by human experts but the scale of modern proteomics experiments makes this impractical. In computer science, expert systems are a mature technology to implement a list of rules generated by interviews with practitioners. We here develop such an expert system, making use of literature knowledge as well as a large body of high mass accuracy and pure fragmentation spectra. Interestingly, we find that even with high mass accuracy data, rule sets can quickly become too complex, leading to over-annotation. Therefore we establish a rigorous false discovery rate, calculated by random insertion of peaks from a large collection of other MS/MS spectra, and use it to develop an optimized knowledge base. This rule set correctly annotates almost all peaks of medium or high abundance. For high resolution HCD data, median intensity coverage of fragment peaks in MS/MS spectra increases from 58% by search engine annotation alone to 86%. The resulting annotation performance surpasses a human expert, especially on complex spectra such as those of larger phosphorylated peptides. Our system is also applicable to high resolution CID data. It is available both as a part of MaxQuant and via a webserver that only requires an MS/MS spectrum and the corresponding peptides sequence, and which outputs publication quality, annotated MS/MS spectra (www.biochem.mpg.de/mann/tools/). It provides expert knowledge to beginners in the field of MS-based proteomics and helps expert users to focus on unusual and possibly novel types of fragment ions.

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Thematic Minireview Series on Circular Proteins

Thematic Minireview Series on Circular Proteins | proteomics | Scoop.it

Circular proteins have now been discovered in all kingdoms of life and are characterized by their exceptional stability and the diversity of their biological activities, primarily in the realm of host defense functions. This thematic minireview series provides an overview of the distribution, evolution, activities, and biological synthesis of circular proteins. It also reviews approaches that biological chemists are taking to develop synthetic methods for making circular proteins in the laboratory. These approaches include solid-phase peptide synthesis based on an adaption of native chemical ligation technology and recombinant DNA approaches that are amenable to the in-cell production of cyclic peptide libraries. The thioester-mediated native chemical ligation approach mimics, to some extent, elements of the natural biosynthetic reaction, which, for disulfide-rich cyclic peptides, appears to involve asparaginyl endopeptidase-mediated processing from larger precursor proteins.

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