Confidence In Protein Identification Research Articles

Improving Protein Identification Using Complementary Fragmentation Techniques in Fourier Transform Mass Spectrometry

Identification of proteins by MS/MS is performed by matching experimental mass spectra against calculated spectra of all possible peptides in a protein data base. The search engine assigns each spectrum a score indicating how well the experimental data complies with the expected one; a higher score means increased confidence in the identification. One problem is the false-positive identifications, which arise from incomplete data as well as from the presence of misleading ions in experimental mass spectra due to gas-phase reactions, stray ions, contaminants, and electronic noise. We employed a novel technique of reduction of false positives that is based on a combined use of orthogonal fragmentation techniques electron capture dissociation (ECD) and collisionally activated dissociation (CAD). Since ECD and CAD exhibit many complementary properties, their combined use greatly increased the analysis specificity, which was further strengthened by the high mass accuracy (approximately 1 ppm) afforded by Fourier transform mass spectrometry. The utility of this approach is demonstrated on a whole cell lysate from Escherichia coli. Analysis was made using the data-dependent acquisition mode. Extraction of complementary sequence information was performed prior to data base search using in-house written software. Only masses involved in complementary pairs in the MS/MS spectrum from the same or orthogonal fragmentation techniques were submitted to the data base search. ECD/CAD identified twice as many proteins at a fixed statistically significant confidence level with on average a 64% higher Mascot score. The confidence in protein identification was hereby increased by more than 1 order of magnitude. The combined ECD/CAD searches were on average 20% faster than CAD-only searches. A specially developed test with scrambled MS/MS data revealed that the amount of false-positive identifications was dramatically reduced by the combined use of CAD and ECD.

Quantitative Proteome Analysis of Human Plasma following in Vivo Lipopolysaccharide Administration Using 16O/18O Labeling and the Accurate Mass and Time Tag Approach

Identification of novel diagnostic or therapeutic biomarkers from human blood plasma would benefit significantly from quantitative measurements of the proteome constituents over a range of physiological conditions. Herein we describe an initial demonstration of proteome-wide quantitative analysis of human plasma. The approach utilizes postdigestion trypsin-catalyzed 16O/18O peptide labeling, two-dimensional LC-FTICR mass spectrometry, and the accurate mass and time (AMT) tag strategy to identify and quantify peptides/proteins from complex samples. A peptide accurate mass and LC elution time AMT tag data base was initially generated using MS/MS following extensive multidimensional LC separations to provide the basis for subsequent peptide identifications. The AMT tag data base contains >8,000 putative identified peptides, providing 938 confident plasma protein identifications. The quantitative approach was applied without depletion of high abundance proteins for comparative analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Accurate quantification of changes in protein abundance was demonstrated by both 1:1 labeling of control plasma and the comparison between the plasma samples following LPS administration. A total of 429 distinct plasma proteins were quantified from the comparative analyses, and the protein abundances for 25 proteins, including several known inflammatory response mediators, were observed to change significantly following LPS administration.

Analysis of Shotgun Proteomics and RNA Profiling Data from Arabidopsis thaliana Chloroplasts

The integration of data from transcriptional profiling and shotgun proteomics experiments provides additional information about the identified proteins that goes beyond their plain detection. We have analyzed results from MS/MS shotgun detection of 426 Arabidopsis chloroplast proteins and genome-wide RNA profiling to identify correlations between gene expression, protein abundance and protein characteristics that influence their detection in high-throughput proteome analyses. The integrated data analysis revealed a significant molecular mass bias for the detection of proteins that were expressed at low transcript levels. Overall, the sequence coverage of most of the identified proteins increases with transcript levels indicating a positive correlation between transcript and relative protein abundance. This does not apply to a subset of the identified proteins suggesting specific properties that alter their detection in shotgun proteomics. This integrative comparison is a suitable strategy to validate large scale proteomics data and offers an assessment of the depth of the proteome analysis and the confidence in protein identification.

Efficient Micro-Recovery and Guanidination of Peptides Directly from MALDI Target Spots

A method is presented for the recovery and subsequent guanidination of tryptic peptides from samples previously spotted on a matrix-assisted laser desorption/ionization (MALDI) target. The procedure is shown to have applicability to both in-solution and in-gel digests, yielding improved confidence in protein identification and sequence coverage in all instances. Recovery from the plate is essentially quantitative, with no residual analyte observed on the target spot. The technique is rapid, simple, and has extended applicability to other processing steps, including (but not limited to) derivatization for specific peptide studies or enzymatic treatment for subsequent profiling of posttranslational modifications. This method circumvents the failure of an initial analysis to generate suitable information and is particularly relevant for the analysis of precious samples.

Improved mass accuracy for tandem mass spectrometry

With the emergence of top-down proteomics, the ability to achieve high mass measurement accuracy on tandem MS/MS data will be beneficial for protein identification and characterization. (FT-ICR) Fourier transform ion cyclotron resonance mass spectrometers are the ideal instruments to perform these experiments with their ability to provide high resolution and mass accuracy. A major limitation to mass measurement accuracy in FT-ICR instruments arises from the occurrence of space charge effects. These space charge effects shift the cyclotron frequency of the ions, which compromises the mass measurement accuracy. While several methods have been developed that correct these space charge effects, they have limitations when applied to MS/MS experiments. It has already been shown that additional information inherent in electrospray spectra can be used for improved mass measurement accuracy with the use of a computer algorithm called DeCAL (deconvolution of Coulombic affected linearity). This paper highlights a new application of the strategy for improved mass accuracy in tandem mass analysis. The results show a significant improvement in mass measurement accuracy on complex electron capture dissociation spectra of proteins. We also demonstrate how the improvement in mass accuracy can increase the confidence in protein identification from the fragment masses of proteins acquired in MS/MS experiments.

Automated Protein Identification by the Combination of MALDI MS and MS/MS Spectra from Different Instruments

The identification of proteins separated on two-dimensional gels is most commonly performed by trypsin digestion and subsequent matrix-assisted laser desorption ionization (MALDI) with time-of-flight (TOF). Recently, atmospheric pressure (AP) MALDI coupled to an ion trap (IT) has emerged as a convenient method to obtain tandem mass spectra (MS/MS) from samples on MALDI target plates. In the present work, we investigated the feasibility of using the two methodologies in line as a standard method for protein identification. In this setup, the high mass accuracy MALDI-TOF spectra are used to calibrate the peptide precursor masses in the lower mass accuracy AP-MALDI-IT MS/MS spectra. Several software tools were developed to automate the analysis process. Two sets of MALDI samples, consisting of 142 and 421 gel spots, respectively, were analyzed in a highly automated manner. In the first set, the protein identification rate increased from 61% for MALDI-TOF only to 85% for MALDI-TOF combined with AP-MALDI-IT. In the second data set the increase in protein identification rate was from 44% to 58%. AP-MALDI-IT MS/MS spectra were in general less effective than the MALDI-TOF spectra for protein identification, but the combination of the two methods clearly enhanced the confidence in protein identification.

Depth of Proteome Issues: A Yeast Isotope-Coded Affinity Tag Reagent Study

As a test case for optimizing how to perform proteomics experiments, we chose a yeast model system in which the UPF1 gene, a protein involved in nonsense-mediated mRNA decay, was knocked out by homologous recombination. The results from five complete isotope-coded affinity tag (ICAT) experiments were combined, two using matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) and three using electrospray MS/MS. We sought to assess the reproducibility of peptide identification and to develop an informatics structure that characterizes the identification process as well as possible, especially with regard to tenuous identifications. The cleavable form of the ICAT reagent system was used for quantification. Most proteins did not change significantly in expression as a consequence of the upf1 knockout. As expected, the Upf1 protein itself was down-regulated, and there were reproducible increases in expression of proteins involved in arginine biosynthesis. Initially, it seemed that about 10% of the proteins had changed in expression level, but after more thorough examination of the data it turned out that most of these apparent changes could be explained by artifacts of quantification caused by overlapping heavy/light pairs. About 700 proteins altogether were identified with high confidence and quantified. Many peptides with chemical modifications were identified, as well as peptides with noncanonical tryptic termini. Nearly all of these modified peptides corresponded to the most abundant yeast proteins, and some would otherwise have been attributed to "single hit" proteins at low confidence. To improve our confidence in the identifications, in MALDI experiments, the parent masses for the peptides were calibrated against nearby components. In addition, five novel parameters reflecting different aspects of identification were collected for each spectrum in addition to the Mascot score that was originally used. The interrelationship between these scoring parameters and confidence in protein identification is discussed.

Effect of ionization mode in the analysis of proteolytic protein digests

The bottom-up sequencing approach relies on an accurate mass fingerprint of the peptide fragments produced from enzymatic digestion. However, this technique is complicated by “dirty” mass spectra due to matrix interferences and, especially with regard to electrospray ionization data, the presence of multiply-charged species, both of which produce false and incorrect hits in database searches. Here, it is shown that the quality of mass spectra of peptide digests can be improved significantly by changing the polarity of the mass spectrometer’s ionization source. Using the negative ion mode in both matrix-assisted laser desorption ionization and electrospray ionization of peptide digests results in the detection of a number of peaks in the mass spectra that are not observed in positive ion mode. In addition, there is considerable reduction in interference from contaminant species in the negative ion mode versus the positive ion mode. By combining the positive ion and negative ion mode data, greater sequence coverage and improved database search scores were obtained. When digestion is incomplete, a dramatic enrichment in the observed abundances for digest product peptides versus the intact proteins could be obtained by changing the pH and the ionization mode. The present results highlight the importance of using positive ion mode and negative ion mode combined to improve overall amino acid sequence coverage and improve confidence in protein identification.

Development of an LC-MALDI method for the analysis of protein complexes

In this study, a two-dimensional LC-MALDI-TOF/TOF method has been developed for analyzing protein complexes. In our hands, the method has proven to be an excellent strategy for the analysis of protein complexes isolated in pull-down experiments. This is in part because the preservation of the chromatographic separation on a MALDI target yields an “unlimited” amount of time to obtain MS/MS spectra, making it possible to probe more deeply into complex samples. A brief statistical analysis was performed on the data obtained from the LC-MALDI-TOF/TOF system in order to better understand peptide fragmentation patterns under high-energy collision conditions. These statistical analyses provided some insight into how to evaluate the quality and accuracy of the database search results derived from the TOF/TOF-based analysis. The potential of the method was demonstrated by the successful identification of all the known penicillin-binding proteins in E. coli isolated using a drug-based pull-down with ampicillin as the bait. The performance of the LC-MALDI-TOF/TOF system was compared with that of an equivalent 2D LC-ESI-MS/MS approach, in the analysis of a protein bait-based pull-down. Regardless of the number of peptides identified in the ESI versus MALDI approach, the two approaches were found to be complementary. When the data is merged at the peptide level, the combined result gives higher Mascot scores and an overall higher confidence in protein identification than with either approach alone.

Interpretation of mass spectrometry data for high-throughput proteomics.

Recent developments in proteomics have revealed a bottleneck in bioinformatics: high-quality interpretation of acquired MS data. The ability to generate thousands of MS spectra per day, and the demand for this, makes manual methods inadequate for analysis and underlines the need to transfer the advanced capabilities of an expert human user into sophisticated MS interpretation algorithms. The identification rate in current high-throughput proteomics studies is not only a matter of instrumentation. We present software for high-throughput PMF identification, which enables robust and confident protein identification at higher rates. This has been achieved by automated calibration, peak rejection, and use of a meta search approach which employs various PMF search engines. The automatic calibration consists of a dynamic, spectral information-dependent algorithm, which combines various known calibration methods and iteratively establishes an optimised calibration. The peak rejection algorithm filters signals that are unrelated to the analysed protein by use of automatically generated and dataset-dependent exclusion lists. In the "meta search" several known PMF search engines are triggered and their results are merged by use of a meta score. The significance of the meta score was assessed by simulation of PMF identification with 10,000 artificial spectra resembling a data situation close to the measured dataset. By means of this simulation the meta score is linked to expectation values as a statistical measure. The presented software is part of the proteome database ProteinScape which links the information derived from MS data to other relevant proteomics data. We demonstrate the performance of the presented system with MS data from 1891 PMF spectra. As a result of automatic calibration and peak rejection the identification rate increased from 6% to 44%.

Optimization of in-gel protein digestion system in combination with thin-gel separation and negative staining in 96-well plate format.

Improvement of in-gel digestion efficiency is highly desirable for one- or two-dimensional gel electrophoretic separation and mass spectrometric (MS) analysis in proteomics, because the resultant increases in sequence coverage and MS signal intensity lead to higher confidence in protein identification. Here an optimized in-gel digestion system, in combination with thin-gel separation and negative staining in a high-throughput format using 96-well plates, is described. The combination of negative staining and protein separation on a 0.9 mm thick gel showed a clear improvement in in-gel digestion efficiency in comparison with the more typical protocols such as the combination of silver staining and a 1.0 mm gel. In addition, the use of 96-well plates to increase throughput did not decrease the efficiency of this strategy when the stirring of the gel pieces in processes such as destaining, washing, gel-shrinking and peptide extraction was performed by sonication instead of shaking the plates. This procedure was optimized and applied to identify proteins of the postsynaptic density fraction; 105 proteins were identified after SDS-PAGE separation.

The Tools of Proteomics

Publisher Summary The chapter focuses on the tools used in proteomics. Mass spectrometry (MS)-based methods have become the primary technology used to identify proteins. The sensitivity of MS instrumentation allows for the identification of proteins below the 1-pmol level and in many cases in the femtomole or even the attomole range. The mass-measurement accuracy afforded with current MS technology, routinely to less than 50 parts per million (ppm) and better, provides more confident protein identifications. Additional sequence information provided by tandem MS (MS/MS) approaches improves the level of confidence of the identification. The major technological advances associated with MS are described in this chapter along with the MS-based methods related to the analysis of proteins separated by one- and two-dimensional gel electrophoresis. Non-polyacrylamide gel electrophoresis (PAGE)-based analyses are also addressed; however, separation methods such as two-dimensional liquid chromatography have specific advantages over the PAGE approach. The developments can be divided into a few general categories, including ionization methods, analyzers, and sample processing. A vast majority of protein identifications made using MS data is accomplished through a comparison with known protein sequences found in a variety of different databases populated with either protein or DNA sequence information. The chapter focuses on the most commonly used software for identifying proteins both through peptide mapping and fragmentation. Peptide mapping is the simplest method for identifying a protein. The second common method to identify a protein through characterization at the peptide level is by tandem MS, which produces fragmentation spectra. In addition, individual laboratories have also designed tools to mine their own data in an attempt to maximize the amount of information retrievable from a proteomic analysis.

Probability-based validation of protein identifications using a modified SEQUEST algorithm.

Database-searching algorithms compatible with shotgun proteomics match a peptide tandem mass spectrum to a predicted mass spectrum for an amino acid sequence within a database. SEQUEST is one of the most common software algorithms used for the analysis of peptide tandem mass spectra by using a cross-correlation (XCorr) scoring routine to match tandem mass spectra to model spectra derived from peptide sequences. To assess a match, SEQUEST uses the difference between the first- and second-ranked sequences (ACn). This value is dependent on the database size, search parameters, and sequence homologies. In this report, we demonstrate the use of a scoring routine (SEQUEST-NORM) that normalizes XCorr values to be independent of peptide size and the database used to perform the search. This new scoring routine is used to objectively calculate the percent confidence of protein identifications and posttranslational modifications based solely on the XCorr value.

Advanced mass spectrometric methods for the rapid and quantitative characterization of proteomes

Progress is reviewed towards the development of a global strategy that aims to extend the sensitivity, dynamic range, comprehensiveness and throughput of proteomic measurements based upon the use of high performance separations and mass spectrometry. The approach uses high accuracy mass measurements from Fourier transform ion cyclotron resonance mass spectrometry (FTICR) to validate peptide ‘accurate mass tags’ (AMTs) produced by global protein enzymatic digestions for a specific organism, tissue or cell type from ‘potential mass tags’ tentatively identified using conventional tandem mass spectrometry (MS/MS). This provides the basis for subsequent measurements without the need for MS/ MS. High resolution capillary liquid chromatography separations combined with high sensitivity, and high resolution accurate FTICR measurements are shown to be capable of characterizing peptide mixtures of more than 105 components. The strategy has been initially demonstrated using the microorganisms Saccharomyces cerevisiae and Deinococcus radiodurans. Advantages of the approach include the high confidence of protein identification, its broad proteome coverage, high sensitivity, and the capability for stableisotope labeling methods for precise relative protein abundance measurements. Abbreviations: LC, liquid chromatography; FTICR, Fourier transform ion cyclotron resonance; AMT, accurate mass tag; PMT, potential mass tag; MMA, mass measurement accuracy; MS, mass spectrometry; MS/MS, tandem mass spectrometry; ppm, parts per million.

The use of accurate mass tags for high-throughput microbial proteomics.

We describe and review progress towards a global strategy that aims to extend the sensitivity, dynamic range, comprehensiveness, and throughput of proteomic measurements for microbial systems based upon the use of polypeptide accurate mass tags (AMTs) produced by global protein enzymatic digestions. The two-stage strategy exploits high accuracy mass measurements using Fourier transform ion cyclotron resonance mass spectrometry (FTICR) to validate polypeptide AMTs for a specific organism, from potential mass tags tentatively identified using tandem mass spectrometry (MS/MS), providing the basis for subsequent measurements without the need for routine MS/MS. A high-resolution capillary liquid chromatography separation combined with high sensitivity, and high-resolution accurate FTICR measurements is shown to be capable of characterizing polypeptide mixtures of more than 10(5) components, sufficient for broad protein identification using AMTs. Advantages of the approach include the high confidence of protein identification, its broad proteome coverage, and the capability for stable-isotope labeling methods for precise relative protein abundance measurements. The strategy has been initially evaluated using the microorganisms Saccharomyces cerevisiae and Deinococcus radiodurans. Additional developments, including the use of multiplexed-MS/MS capabilities and methods for dynamic range expansion of proteome measurements that promise to further extend the quality of proteomics measurements, are also described.

Investigation of the applicability of a sequential digestion protocol using trypsin and leucine aminopeptidase M for protein identification by matrix-assisted laser desorption/ionization--time of flight mass spectrometry.

An investigation of the applicability of a sequential digestion procedure involving endo- and exoprotease digestion of proteins is reported. The procedure involves the digestion of a protein sample with trypsin, yielding peptide fragments characteristic of the protein. The resulting mixture of peptide fragments is then subjected to N-terminal sequencing with leucine aminopeptidase M (LAP), with matrix-assisted laser desorption ionization-time of flight mass spectrometric analysis of the various digestion products. Several proteins in solution, as well as gel extracted proteins were subjected to this sequential enzyme digestion procedure. The results of these experiments reveal that LAP will preferentially cleave specific peptides of the trypsin digested sample with high efficiency, while leaving other peptides undigested. Also, the length of the amino acid sequence tags that can be generated with this method is limited; the longest sequence tag generated from a single tryptic peptide was four amino acids, even though the digestion was allowed to proceed for long times. In the experiments, N-terminal digestion products were detected as early as two minutes, or as late as 90 minutes, following the addition of LAP to the sample. The method was shown to be effective for subpicomole starting quantities of protein, although with some loss in digestion efficiency at lower concentrations of starting material. This method is useful in providing additional sequence information to increase the level of confidence in protein identification, as illustrated in the identification of bacterial proteins fractionated by high-performance liquid chromatography. In some instances, this method can provide additional sequence information where post source decay and nanospray mass spectrometry failed to generate fragment ion spectra. This is illustrated in an example where the procedure was applied to a membrane protein, CD9, that had been isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Although the sequential digestion procedure requires more human intervention, it is a straightforward method and can be readily implemented.

Analysis of protein sequences and protein complexes by matrix-assisted laser desorption/ionization mass spectrometry

In the context of proteome analysis, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) can fulfil the two tasks of primary structure verification and protein identification. As an illustration of the first of these tasks, the sequence of Eschericha coli isoleucyl-tRNA synthetase, a protein with 15 reported sequence conflicts, has been established by means of MALDI mass mapping. The identification of mitochondrial proteins participating in a yeast supramolecular complex exhibiting NADH dehydrogenase activity highlights the performances of MALDI-MS for the second task. The spectral suppression phenomenon occurring for complex peptide mixtures analysed by MALDI is discussed, as well as the role of post-source decay analysis for confident protein identification.

Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

High-sensitivity, high-throughput analysis of proteins for proteomics studies is usually performed by polyacrylamide gel electrophoresis in combination with mass spectrometry. However, the quality of the data obtained depends on the in-gel digestion procedure employed. This work describes an improvement in the in-gel digestion efficiency for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis. A dramatic improvement in the coverage of tryptic peptides was observed when n-octyl glucoside was added to the buffer. Whole cell extracted proteins from S. cerevisiae were separated by two-dimensional gel electrophoresis and stained with silver. Protein spots were identified using our improved in-gel digestion method and MALDI-TOFMS. In addition, the mass spectra obtained by using the matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) were compared with those obtained using 2,5-dihydroxybenzoic acid (DHB). The DHB matrix usually gave more peaks, which led to higher sequence coverage and, consequently, to higher confidence in protein identification. This improved in-gel digestion protocol is simple and useful for protein identification by MALDI-TOFMS.

Investigation of the Applicability of a Sequential Digestion Protocol Using Trypsin and Leucine Aminopeptidase M for Protein Identification by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

An investigation into the applicability of a sequential digestion procedure involving endo- and exoprotease digestion of proteins is reported. The procedure involves the digestion of a protein sample with trypsin, yielding peptide fragments characteristic of the protein. The resulting mixture of peptide fragments is then subjected to N-terminal sequencing with leucine aminopeptidase M (LAP), with matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of the various digestion products. Several proteins in solution, as well as gel-extracted proteins, were subjected to this sequential enzyme digestion procedure. The results of these experiments reveal that LAP will preferentially cleave specific peptides of the trypsin-digested sample with high efficiency, while leaving other peptides undigested. Also, the length of the amino acid sequence tags that can be generated with this method is limited; the longest sequence tag generated from a single tryptic peptide was four amino acids, even though the digestion was allowed to proceed for long times. In the experiments, N-terminal digestion products were detected as early as two minutes, or as late as 90 minutes, following the addition of LAP to the sample. The method was shown to be effective for sub-picomole starting quantities of protein, although with some loss in digestion efficiency at lower concentrations of starting material. This method is useful in providing additional sequence information to increase the level of confidence in protein identification, as illustrated in the identification of bacterial proteins fractionated by HPLC. In some instances, this method can provide additional sequence information where post-source decay and nanospray mass spectrometry failed to generate fragment-ion spectra. This is illustrated by an example where the procedure was applied to a membrane protein, CD9, that had been isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although the sequential digestion procedure requires more human intervention, it is a straightforward method and can be readily implemented.

Analysis of Protein Sequences and Protein Complexes by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

In the context of proteome analysis, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry can fulfil the two tasks of primary structure verification and protein identification. As an illustration of the first of these tasks, the sequence of E. coli isoleucyl-tRNA synthetase, a protein with 15 reported sequence conflicts, has been established by means of MALDI mass mapping. The identification of mitochondrial proteins participating in a yeast supramolecular complex exhibiting NADH dehydrogenase activity highlights the performance of MALDI mass spectrometry for the second task. The spectral suppression phenomenon occurring for complex peptide mixtures analyzed by MALDI is discussed, as well as the role of post-source decay (PSD) analysis for confident protein identification.