Two‐step OFFGEL approach for effective peptide separation compatible with iTRAQ labeling

Shotgun proteomic analyses are increasingly becoming methods of choice for complex samples. The development of effective methods for fractionating peptides to reduce the complexity of the sample before mass analysis is a key point in this strategy. The OFFGEL technology has recently become a tool of choice in proteomic analysis at peptide level. This OFFGEL electrophoresis (OGE) approach allows the in‐solution separation of peptides from various biological sources by isoelectric focusing in highly resolved 24 fractions. It was also demonstrated that OGE technology is a filtering tool for pI‐based validation of peptide identification. As peptide OGE is compatible with iTRAQ labeling, OGE is finding valuable applications in quantitative proteomics as well. The aim of this study is to explain a new 2D‐OGE approach that improves the proteomic coverage of complex mixtures such as colorectal cell line lysates, and which is compatible with iTRAQ labeling.     

ProteoMod: A new tool to quantitate protein post-translational modifications

Post-translational modifications (PTMs) are known to regulate biological processes by controlling protein function. The effect of a PTM on protein function depends critically on the position and the number of modifications. While there are convenient methods available to qualitatively examine modifications like phosphorylation, glycosylation, acetylation and methylation, methods available for their quantitative assessment are cumbersome. We have developed a new tool that allows quantitation of the number of phosphorylation events in proteins with ease. The "ProteoMod" tool depends on shifts in the isoelectric points of proteins upon post-translational change. The extent of shift exhibited upon phosphorylation is algorithmically converted into the number of phosphorylations conferred. The validity of ProteoMod was confirmed by examining proteins with previously known number of phosphorylations. The list of proteins examined included HSP27, HSP70 and tumor suppressor p53. The approach can also be applied to estimate modifications like acetylation, methylation and sialylation in proteins. We analyzed shifts in isoelectric points due to sialylation events in N-glycoproteins. Using influenza hemagglutinin we show that shifts in isoelectric points correlate with intracellular distribution of this model membrane protein. In addition to extending the application of two dimensional gel electrophoresis to quantitate modifications, our study also highlights its potential use in cell biology.     

Added value for tandem mass spectrometry shotgun proteomics data validation through isoelectric focusing of peptides

A very popular approach in proteomics is the so-called "shotgun LC-MS/MS" strategy. In its mostly used form, a total protein digest is separated by ion exchange fractionation in the first dimension followed by off- or on-line RP LC-MS/MS. We replaced the first dimension by isoelectric focusing in the liquid phase using the Off-Gel device producing 15 fractions. As peptides are separated by their isoelectric point in the first dimension and hydrophobicity in the second, those experimentally derived parameters (pI and R(T)) can be used for the validation of potentially identified peptides. We applied this strategy to a cellular extract of Drosophila Kc167 cells and identified peptides with two different database search engines, namely PHENYX and SEQUEST, with PeptideProphet validation of the SEQUEST results. PHENYX returned 7582 potential peptide identifications and SEQUEST 7629. The SEQUEST results were reduced to 2006 identifications by validation with PeptideProphet. Validation of the PeptideProphet, SEQUEST and PHENYX results by pI and R(T) parameters confirmed 1837 PeptideProphet identifications while in the remainder of the SEQUEST results another 1130 peptides were found to be likely hits. The validation on PHENYX resulted in the fixation of a solid p-value threshold of <1 x 10(-04) that sets by itself the correct identification confidence to >95%, and a final count of 2034 highly confident peptide identifications was achieved after pI and R(T) validation. Although the PeptideProphet and PHENYX datasets have a very high confidence the overlap of common identifications was only at 79.4%, to be explained by the fact that data interpretation was done searching different protein databases with two search engines of different algorithms. The approach used in this study allowed for an automated and improved data validation process for shotgun proteomics projects producing MS/MS peptide identification results of very high confidence.     

Reference map for liquid chromatography–mass spectrometry-based quantitative proteomics

The accurate mass and time (AMT) tag strategy has been recognized as a powerful tool for high-throughput analysis in liquid chromatography–mass spectrometry (LC–MS)-based proteomics. Due to the complexity of the human proteome, this strategy requires highly accurate mass measurements for confident identifications. We have developed a method of building a reference map that allows relaxed criteria for mass errors yet delivers high confidence for peptide identifications. The samples used for generating the peptide database were produced by collecting cysteine-containing peptides from T47D cells and then fractionating the peptides using strong cationic exchange chromatography (SCX). LC–tandem mass spectrometry (MS/MS) data from the SCX fractions were combined to create a comprehensive reference map. After the reference map was built, it was possible to skip the SCX step in further proteomic analyses. We found that the reference-driven identification increases the overall throughput and proteomic coverage by identifying peptides with low intensity or complex interference. The use of the reference map also facilitates the quantitation process by allowing extraction of peptide intensities of interest and incorporating models of theoretical isotope distribution.     

MS-Simulator: Predicting Y-Ion Intensities for Peptides with Two Charges Based on the Intensity Ratio of Neighboring Ions

For the identification of peptides with tandem mass spectrometry (MS/MS), many software tools rely on the comparison between an experimental spectrum and a theoretically predicted spectrum. Consequently, the accurate prediction of the theoretical spectrum from a peptide sequence can potentially improve the peptide identification performance and is an important problem for mass spectrometry based proteomics. In this study a new approach, called MS-Simulator, is presented for predicting the y-ion intensities in the spectrum of a given peptide. The new approach focuses on the accurate prediction of the relative intensity ratio between every two adjacent y-ions. The theoretical spectrum can then be derived from these ratios. The prediction of a ratio is a closed-form equation that involves up to five consecutive amino acids nearby the two y-ions and the two peptide termini. Compared with another existing spectrum prediction tool MassAnalyzer, the new approach not only simplifies the computation, but also improves the prediction accuracy.     

Enhanced N-glycosylation site exploitation of sialoglycopeptides by peptide IPG-IEF assisted TiO2 chromatography

Playing an important role in a broad range of biological and pathological processes, sialylation has been drawing wide interest. The efficient sialoglycopeptides enrichment methods are therefore attracting considerable attention. In this paper, we first compared two conventional enrichment methods, lectin and TiO(2), and analyzed their characteristics. Furthermore, considering the highly negatively charged nature of sialic acids, we developed a new strategy, peptide immobilized pH gradient isoelectric focusing (IPG-IEF) assisted TiO(2) chromatography (PIAT), for the highly efficient enrichment of sialoglycopeptides. In this method, peptides were first separated into 24 fractions using peptide IPG-IEF. Sialoglycopeptides were relatively concentrated in low-pH fractions of the immobilized pH strips and were captured using TiO(2) chromatography. As a result, 614?N-glycosylation sites were identified in 582 sialoglycopeptides within 322 sialoglycoproteins from rat liver using PIAT. To our knowledge, this work represents one of the most comprehensive sialoglycoproteomic analyses in general and exhibits the largest database of sialoglycoproteome in rat liver currently. So the new strategy introduced here exhibits high efficiency and universality in the sialoglycopeptide enrichment, and is a powerful tool for sialoglycoproteome exploration.     

PeaksPTM: Mass spectrometry-based identification of peptides with unspecified modifications

Tandem mass spectrometry (MS/MS) has been routinely used to identify peptides from a protein sequence database. To identify post-translationally modified peptides, most existing software requires the specification of a few possible modifications. However, such knowledge of possible modifications is not always available. In this paper, we describe a new algorithm for identifying modified peptides without requiring the user to specify the possible modifications; instead, all modifications from the Unimod database are considered. Meanwhile, several new techniques are employed to avoid the exponential growth of the search space, as well as to control the false discoveries due to this unrestricted search approach. Finally, a software tool, PeaksPTM, has been developed and already achieved a stronger performance than competitive tools for unrestricted identification of post-translational modifications.     

Proteomic analysis of colorectal cancer: prefractionation strategies using two-dimensional free-flow electrophoresis

This review deals with the application of a new prefractionation tool, free-flow electrophoresis (FFE), for proteomic analysis of colorectal cancer (CRC). CRC is a leading cause of cancer death in the Western world. Early detection is the single most important factor influencing outcome of CRC patients. If identified while the disease is still localized, CRC is treatable. To improve outcomes for CRC patients there is a pressing need to identify biomarkers for early detection (diagnostic markers), prognosis (prognostic indicators), tumour responses (predictive markers) and disease recurrence (monitoring markers). Despite recent advances in the use of genomic analysis for risk assessment, in the area of biomarker identification genomic methods alone have yet to produce reliable candidate markers for CRC. For this reason, attention is being directed towards proteomics as a complementary analytical tool for biomarker identification. Here we describe a proteomics separation tool, which uses a combination of continuous FFE, a liquid-based isoelectric focusing technique, in the first dimension, followed by rapid reversed-phase HPLC (1-6 min/analysis) in the second dimension. We have optimized imaging software to present the FFE/RP-HPLC data in a virtual 2D gel-like format. The advantage of this liquid based fractionation system over traditional gel-based fractionation systems is the ability to fractionate large quantity protein samples. Unlike 2D gels, the method is applicable to both high-M(r) proteins and small peptides, which are difficult to separate, and in the case of peptides, are not retained in standard 2D gels.     

Efficient fractionation and improved protein identification by peptide OFFGEL electrophoresis

The sample fractionation steps conducted prior to mass detection are critically important for the comprehensive analysis of complex protein mixtures. This paper illustrates the effectiveness of OFFGEL electrophoresis with the Agilent 3100 OFFGEL Fractionator for the fractionation of peptides. An Escherichia coli tryptic digest was separated in 24 fractions, and peptides were identified by reversed-phase liquid chromatography on a microfluidic device with mass spectrometric detection. About 90% of the identified individual peptides were found in only one or two fractions. The distribution of the calculated isoelectric points for the peptides identified in each fraction was especially narrow in the acidic pH range. Standard deviations approached the size of the pH segment covered by the respective fraction. The experimental peptide isoelectric point measured by OFFGEL electrophoresis was used as an additional filter for validation of peptide identifications.     

Depletion and fractionation technologies in plasma proteomic analysis

This review intends to survey the traditional and current technologies in the depletion and subfractionation of plasma proteins for further analyses. The value of depletion aims to enrich low-abundant proteins by removing highly abundant proteins, such as albumin or immunoglobulin G, from plasma. With this approach, one can examine both the resulting high- and low-abundant protein fractions. The depleted protein population can be further subfractionated based on their isoelectric point ranges, creating a more discrete pool of proteins for detailed post-translational modification studies by methods such as 2D gel electrophoresis and mass spectrometry. The concept of divide to conquer will greatly enhance our ability to identify and characterize low-abundant proteins and cleaved peptides from plasma as important diagnostic markers or potential drug targets. This can potentially reverse the decline in the development of new plasma diagnostic tests.     

Depletion and fractionation technologies in plasma proteomic analysis

This review intends to survey the traditional and current technologies in the depletion and subfractionation of plasma proteins for further analyses. The value of depletion aims to enrich low-abundant proteins by removing highly abundant proteins, such as albumin or immunoglobulin G, from plasma. With this approach, one can examine both the resulting high- and low-abundant protein fractions. The depleted protein population can be further subfractionated based on their isoelectric point ranges, creating a more discrete pool of proteins for detailed post-translational modification studies by methods such as 2D gel electrophoresis and mass spectrometry. The concept of divide to conquer will greatly enhance our ability to identify and characterize low-abundant proteins and cleaved peptides from plasma as important diagnostic markers or potential drug targets. This can potentially reverse the decline in the development of new plasma diagnostic tests.     

CLPM: A Cross-Linked Peptide Mapping Algorithm for Mass Spectrometric Analysis

Background

Protein-protein, protein-DNA and protein-RNA interactions are of central importance in biological systems. Quadrapole Time-of-flight (Q-TOF) mass spectrometry is a sensitive, promising tool for studying these interactions. Combining this technique with chemical crosslinking, it is possible to identify the sites of interactions within these complexes. Due to the complexities of the mass spectrometric data of crosslinked proteins, new software is required to analyze the resulting products of these studies.

Result

We designed a Cross-Linked Peptide Mapping (CLPM) algorithm which takes advantage of all of the information available in the experiment including the amino acid sequence from each protein, the identity of the crosslinker, the identity of the digesting enzyme, the level of missed cleavage, and possible chemical modifications. The algorithm does in silico digestion and crosslinking, calculates all possible mass values and matches the theoretical data to the actual experimental data provided by the mass spectrometry analysis to identify the crosslinked peptides.

Conclusion

Identifying peptides by their masses can be an efficient starting point for direct sequence confirmation. The CLPM algorithm provides a powerful tool in identifying these potential interaction sites in combination with chemical crosslinking and mass spectrometry. Through this cost-effective approach, subsequent efforts can quickly focus attention on investigating these specific interaction sites.

     

GMOseek: a user friendly tool for optimized GMO testing

Background

With the increasing pace of new Genetically Modified Organisms (GMOs) authorized or in pipeline for commercialization worldwide, the task of the laboratories in charge to test the compliance of food, feed or seed samples with their relevant regulations became difficult and costly. Many of them have already adopted the so called "matrix approach" to rationalize the resources and efforts used to increase their efficiency within a limited budget. Most of the time, the "matrix approach" is implemented using limited information and some proprietary (if any) computational tool to efficiently use the available data.

Results

The developed GMOseek software is designed to support decision making in all the phases of routine GMO laboratory testing, including the interpretation of wet-lab results. The tool makes use of a tabulated matrix of GM events and their genetic elements, of the laboratory analysis history and the available information about the sample at hand. The tool uses an optimization approach to suggest the most suited screening assays for the given sample. The practical GMOseek user interface allows the user to customize the search for a cost-efficient combination of screening assays to be employed on a given sample. It further guides the user to select appropriate analyses to determine the presence of individual GM events in the analyzed sample, and it helps taking a final decision regarding the GMO composition in the sample. GMOseek can also be used to evaluate new, previously unused GMO screening targets and to estimate the profitability of developing new GMO screening methods.

Conclusion

The presented freely available software tool offers the GMO testing laboratories the possibility to select combinations of assays (e.g. quantitative real-time PCR tests) needed for their task, by allowing the expert to express his/her preferences in terms of multiplexing and cost. The utility of GMOseek is exemplified by analyzing selected food, feed and seed samples from a national reference laboratory for GMO testing and by comparing its performance to existing tools which use the matrix approach. GMOseek proves superior when tested on real samples in terms of GMO coverage and cost efficiency of its screening strategies, including its capacity of simple interpretation of the testing results.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-258) contains supplementary material, which is available to authorized users.     

The proteomic analysis improved by cleavage kinetics‐based fractionation of tryptic peptides

Selective enrichment of specific peptides is an effective way to identify low abundance proteins. Fractionation of peptides prior to mass spectrometry is another widely used approach to reduce sample complexity in order to improve proteome coverage.In this study, we designed a multi‐stage digestion strategy to generate peptides with different trypsin cleavage kinetics. It was found that each of the collected peptide fractions yielded many new protein identifications compared to the control group due to the reduced complexity. The overlapping peptides identified between adjacent fractions were very low, indicating that each fraction had different sets of peptides. The multi‐stage digestion strategy separates tryptic peptides with different cleavage kinetics while RPLC separates peptides with different hydrophobicity. These two separation strategies were highly orthogonal, and showed an effective multidimensional separation to improve proteome coverage.     

swissPIT: a novel approach for pipelined analysis of mass spectrometry data

The identification and characterization of peptides from tandem mass spectrometry (MS/MS) data represents a critical aspect of proteomics. Today, tandem MS analysis is often performed by only using a single identification program achieving identification rates between 10-50% (Elias and Gygi, 2007). Beside the development of new analysis tools, recent publications describe also the pipelining of different search programs to increase the identification rate (Hartler et al., 2007; Keller et al., 2005). The Swiss Protein Identification Toolbox (swissPIT) follows this approach, but goes a step further by providing the user an expandable multi-tool platform capable of executing workflows to analyze tandem MS-based data. One of the major problems in proteomics is the absent of standardized workflows to analyze the produced data. This includes the pre-processing part as well as the final identification of peptides and proteins. The main idea of swissPIT is not only the usage of different identification tool in parallel, but also the meaningful concatenation of different identification strategies at the same time. The swissPIT is open source software but we also provide a user-friendly web platform, which demonstrates the capabilities of our software and which is available at http://swisspit.cscs.ch upon request for account.     

Harvest: an open-source tool for the validation and improvement of peptide identification metrics and fragmentation exploration

Background  

Protein identification using mass spectrometry is an important tool in many areas of the life sciences, and in proteomics research in particular. Increasing the number of proteins correctly identified is dependent on the ability to include new knowledge about the mass spectrometry fragmentation process, into computational algorithms designed to separate true matches of peptides to unidentified mass spectra from spurious matches. This discrimination is achieved by computing a function of the various features of the potential match between the observed and theoretical spectra to give a numerical approximation of their similarity. It is these underlying "metrics" that determine the ability of a protein identification package to maximise correct identifications while limiting false discovery rates. There is currently no software available specifically for the simple implementation and analysis of arbitrary novel metrics for peptide matching and for the exploration of fragmentation patterns for a given dataset.     

The amino acid sequence of the polypeptide segment which regulates membrane adhesion (grana stacking) in chloroplasts

A positively charged amino acid sequence, located on the NH2 terminus of the polypeptides of the chlorophyll a/b light harvesting complex, stabilizes thylakoid membrane adhesion. Threonine residues in this segment are the site of light-induced, reversible phosphorylation; this covalent modification results in changes in excitation-energy distribution in chloroplast membranes. Removal of the positively charged peptide by treatment with trypsin or chemical modification of amino acids in the sequence disrupts thylakoid adhesion and inhibits regulation of excitation-energy distribution. Purified preparations of the chlorophyll a/b light harvesting complex consist of 2 major polypeptides of 27 and 26 kDa and 2 minor polypeptides of 29 and 25 kDa (based upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Trypsin treatment of the isolated chlorophyll proteins decreases the apparent molecular mass of the 27- and 26-kDa polypeptides by 1-1.5 kDa and releases 3 peptides; [Lys, Arg], Ser-Ala-Thr-Thr-Lys-Lys, and Ser-Ala-Thr-Thr-Lys. These peptides probably form the overlap sequence, [Lys, Arg]-Ser-Ala-Thr-Thr-Lys-Lys. The polypeptides of the chlorophyll a/b light-harvesting complex were separated by isoelectric focusing into 5 chlorophyll protein fractions which had isoelectric points between 4.0 and 4.55. The 27-kDa polypeptides had an isoelectric point of 4.3, and bound 11 chlorophyll molecules/polypeptide.     

Characterization of low abundant membrane proteins using the protein sequence tag technology

About 25% of open reading frames in fully sequenced genomes are estimated to encode transmembrane proteins that represent valuable targets for drugs. However, the global analysis of membrane proteins has been proven to be problematic, e.g., because of their very amphiphilic nature. In this paper, we show that the recently published Protein Sequence Tag (PST) technology combined with an efficient sample preparation is a powerful method to perform protein analysis of highly enriched membrane fractions. The PST approach is a gel-free proteomics tool for the analysis of proteins, which relies on a "sampling" strategy by isolating N-terminal protein sequence tags from cyanogen bromide cleaved proteins. The identification of these N-terminal PST peptides is based on LC-MS/MS. The effectiveness of the technology is demonstrated for a membrane fraction, which was isolated from crude mitochondria of yeast after alkaline sodium carbonate treatment. The PST approach performed on this fraction analyzed 148 proteins, whereas 84% are identified as membrane proteins. More interestingly, among these membrane proteins 56% are predicted to be of low abundance. These encouraging results are an important step toward the development of a quantitative PST approach (qPST) for the differential display of membrane protein analysis.     

全柱成像毛细管等电聚焦电泳测定艾塞那肽等电点

目的:采用新一代全柱成像毛细管等电聚焦电泳技术(CIEF-WCID)测定艾塞那肽等电点。方法:采用互补性金属氧化物半导体成像技术对样品等电聚焦过程进行实时记录,根据适宜的marker计算得到艾塞那肽的等电点,并对方法的准确度与重复性进行考察。结果:测得艾塞那肽等电点为5.46,与凝胶电泳结果基本一致,相对标准偏差为0.11%。CIEF-WCID方法快速准确,相对误差小于2.5%,重复性良好。结论:CIEF-WCID作为一种新的技术手段可用于艾塞那肽等电点的分析,方法快速、准确、重复性好,可为多肽的质量控制提供一种可靠的分析方法。