An evaluation for cross-species proteomics research by publicly available expressed sequence tag database search using tandem mass spectral data

With 1383 tandem mass spectra derived from 120 individual protein spots separated by the two-dimensional (2-D) gel electrophoresis of protein samples from three different species, comparative analyses were performed by searching the Expressed Sequence Tag (EST) database (DB) and the NCBI non-redundant (nr) DB of green plants, respectively, which uses the Mascot search engine to establish a statistical basis. It was confirmed that the former could identify more peptides manually validated by de novo sequencing (DNS) from fewer species in more closely phylogenetic relationships than the latter in a statistically significant manner. Our data demonstrated that correct peptide identifications were given low Mascot scores (e.g. 6-14) and incorrect peptide identifications were given high Mascot scores (e.g. 68-83). Our data also showed that the current evaluation approaches to protein assignments are unsatisfactory because a few 'false-positive' proteins are recognized and several 'false-negative' proteins are rescued by manual validation.     

Evaluation of confidence and reproducibility in quantitative proteomics performed by a capillary isoelectric focusing-based proteomic platform coupled with a spectral counting approach

Multidimensional separations of the peptides resulting from enzymatic digestions of complex protein mixtures prior to MS/MS, namely shotgun proteomics, is increasingly utilized for large-scale identification and quantitation of proteins. Inherent to the performance of proteomic measurements is the resolving power of each of the separations both separately and in combination. By simply raising the number of CIEF fractions, the resulting enhancement in the overall peak capacity of combined CIEF/nano-RPLC separations greatly reduces the complexity of eluted peptides prior to MS detection and sequencing and increases the proteome coverage. The capabilities of the CIEF-based proteome platform coupled with the spectral counting approach to confidently and reproducibly quantify proteins and changes in protein expression levels among samples are evaluated. Analytical reproducibility of relative protein abundance is determined to exhibit a Pearson R(2) value greater than 0.99 and a CV of 14.1%. The platform is demonstrated to be capable of measuring changes in protein expression as low as 1.5-fold, with confidence following multiple testing adjustment.     

Enrichment of N-terminal sulfonated peptides by a water-soluble fullerene derivative and its applications to highly efficient proteomics

Recent studies have shown that N-terminal sulfonation of tryptic peptides by various sulfonating molecules greatly improves their post-source decay processes (e.g., in matrix-assisted laser desorption ionization) or the gas phase fragmentation processes (e.g., in tandem mass spectrometer), enhancing the ability to identify their sequences de novo. In the present work, we have demonstrated that incorporation of water-soluble C₆₀-N,N-dimethylpyrrolidinium iodide selectively precipitates the 4-sulfophenyl isothiocyanate-modified peptide (SPITC-GGYR, SPITC-ASHLGLAR) by forming a noncovalent ion pair to the SO₃⁻ group of the SPITC, and thereby the C₆₀ derivative can be utilized to enrich the modified peptide. Electrospray ionization (ESI) mass analyses show that the cationic SPITC-GGYR and SPITC-ASHLGLAR species are well separated from unmodified peptides and the modified peptides are subsequently detached from the C₆₀ derivative upon using an acidic solution.     

Applicability of the critical-chromatography concept to proteomics problems: Separation of peptides modeled by a heterogeneous rod

The problems of separation of short peptides are considered in terms of the model of rigid rod adsorption. Analytical expressions relating the retention volume to the sequence of amino-acid residues in the gradient elution are obtained. The model of adsorption of a peptide as a rigid rod is compared with the model of its adsorption as a random-walk chain. The transition of rodlike peptides to the adsorbed state is more abrupt compared with the random-walk chain having the same sequence, while with an increase in the peptide length the random-walk chain model becomes more accurate. It is shown that the model of adsorption of a peptide as a rigid rod for short peptides fits the experimental data and correctly predicts the character of separation of peptides having equal lengths and identical amino-acid compositions but slightly differing in the alternation of residues in a chain.     

On-line counting of cysteine residues in peptides during electrospray ionization by electrogenerated tags and their application to protein identification

The electrochemically induced mass spectrometric tagging of cysteines by substituted hydroquinones was studied for peptides in a classical electrospray solvent (i.e., MeOH/H2O/AcOH 50/49/1). The tagging efficiency was tested with different hydroquinone compounds on an undecapeptide containing one cysteine residue. 2-carboxymethylhydroquinone was the most reactive probe and revealed to be suitable for cysteine quantification in peptides containing up to three cysteine residues, even in the case of two consecutive cysteines in the sequence. We demonstrate the compatibility of the on-line electrochemical tagging method for the cysteine content analysis of peptides coming from gel-free protein digestion procedures. The identification of bovine serum albumin and human alpha-lactalbumin digest samples in a peptide mapping strategy was greatly improved by the application of the electrotagging technique as post-column treatment. Indeed, the determination of cysteine content in the tryptic peptides provided powerful information in order to enhance the identification score as well as the discrimination against other protein candidates. The tagging method was then applied to the determination of four proteins in a model mixture.     

Using intrinsic X-ray absorption spectral differences to identify and map peptides and proteins

The intrinsic variation in the near-edge X-ray absorption fine structure (NEXAFS) spectra of peptides and proteins provide an opportunity to identify and map them in various biological environments, without additional labeling. In principle, with sufficiently accurate spectra, peptides (<50 amino acids) or proteins with unusual sequences (e.g., cysteine- or methionine-rich) should be differentiable from other proteins, since the NEXAFS spectrum of each amino acid is distinct. To evaluate the potential for this approach, we have developed X-SpecSim, a tool for quantitatively predicting the C, N, and O 1s NEXAFS spectra of peptides and proteins from their sequences. Here we present the methodology for predicting such spectra, along with tests of its precision using comparisons to the spectra of various proteins and peptides. The C 1s, N 1s, and O 1s spectra of two novel antimicrobial peptides, Indolicidin (ILPWKWPWWPWRR-NH2) and Sub6 (RWWKIWVIRWWR-NH2), as well as human serum albumin and fibrinogen are reported and interpreted. The ability to identify, differentiate, and quantitatively map an antimicrobial peptide against a background of protein is demonstrated by a scanning transmission X-ray microscopy study of a mixture of albumin and sub6.     

Swarm intelligence based wavelet coefficient feature selection for mass spectral classification: An application to proteomics data

This paper introduces the ant colony algorithm, a novel swarm intelligence based optimization method, to select appropriate wavelet coefficients from mass spectral data as a new feature selection method for ovarian cancer diagnostics. By determining the proper parameters for the ant colony algorithm (ACA) based searching algorithm, we perform the feature searching process for 100 times with the number of selected features fixed at 5. The results of this study show: (1) the classification accuracy based on the five selected wavelet coefficients can reach up to 100% for all the training, validating and independent testing sets; (2) the eight most popular selected wavelet coefficients of the 100 runs can provide 100% accuracy for the training set, 100% accuracy for the validating set, and 98.8% accuracy for the independent testing set, which suggests the robustness and accuracy of the proposed feature selection method; and (3) the mass spectral data corresponding to the eight popular wavelet coefficients can be located by reverse wavelet transformation and these located mass spectral data still maintain high classification accuracies (100% for the training set, 97.6% for the validating set, and 98.8% for the testing set) and also provide sufficient physical and medical meaning for future ovarian cancer mechanism studies. Furthermore, the corresponding mass spectral data (potential biomarkers) are in good agreement with other studies which have used the same sample set. Together these results suggest this feature extraction strategy will benefit the development of intelligent and real-time spectroscopy instrumentation based diagnosis and monitoring systems.     

Optimization and testing of mass spectral library search algorithms for compound identification

Five algorithms proposed in the literature for library search identification of unknown compounds from their low resolution mass spectra were optimized and tested by matching test spectra against reference spectra in the NIST-EPA-NIH Mass Spectral Database. The algorithms were probability-based matching (PBM), dot-product, Hertz et al. similarity index, Euclidean distance, and absolute value distance. The test set consisted of 12,592 alternate spectra of about 8000 compounds represented in the database. Most algorithms were optimized by varying their mass weighting and intensity scaling factors. Rank in the list of candidatc compounds was used as the criterion for accuracy. The best performing algorithm (75% accuracy for rank 1) was the dot-product function that measures the cosine of the angle between spectra represented as vectors. Other methods in order of performance were the Euclidean distance (72%), absolute value distance (68%) PBM (65%), and Hertz et al. (64%). Intensity scaling and mass weighting were important in the optimized algorithms with the square root of the intensity scale nearly optimal and the square or cube the best mass weighting power. Several more complex schemes also were tested, but had little effect on the results. A modest improvement in the performance of the dot-product algorithm was made by adding a term that gave additional weight to relative peak intensities for spectra with many peaks in common.     

A comparative study using liquid scintillation counting and X-ray spectrometry to determine 55Fe in radioactive wastes

The radionuclide ⁵⁵Fe was determined in samples of radioactive wastes from the water cleanup system of the IEA-R1 nuclear research reactor. In order to validate the results, the ⁵⁵Fe activity concentration was measured in eight waste samples and in six simulated samples containing the most important interfering radionuclides. A simple method was employed to separate and purify ⁵⁵Fe from other radionuclides present in these samples, combining co-precipitation with ammonium hydroxide and purification with anionic ion-exchange resin, which enables ⁵⁵Fe to be quantified either by liquid scintillation counting (LSC) or by X-ray spectrometry using a low-energy germanium spectrometer (LEGe). Both measurement methods were used so that the separation and purification process could be confirmed by comparison of spectra with and without the utilization of anionic ion-exchange resin. Activity and interferences were compared in the results obtained from LSC and LEGe measurement methods.     

Part II: defining and quantifying individual and co-cultured intracellular proteomes of two thermophilic microorganisms by GeLC-MS2 and spectral counting

The proteome of extremely thermophilic microorganisms affords a glimpse into the dynamics of microbial ecology of high temperature environments. The secretome, or extracellular proteome of these microorganisms, no doubt harbors technologically important enzymes and other thermostable biomolecules that, to date, have been characterized only to a limited extent. In the first of a two-part study on selected thermophiles, defining the secretome requires a sample preparation method that has no negative impact on all downstream experiments. Following efficient secretome purification, GeLC-MS² analysis and prediction servers suggested probable protein secretion to complement experimental data. In an effort to define the extracellular proteome of the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus, several techniques were considered regarding sample processing to achieve the most in-depth analysis of secreted proteins. Order of operation experiments, all including the C₁₈ bead technique, demonstrated that two levels of sample purification were necessary to effectively desalt the sample and provide sufficient protein identifications. Five sample preparation combinations yielded 71 proteins and the majority described, as enzymatic and putative uncharacterized proteins, anticipate consolidated bioprocessing applications. Nineteen proteins were predicted by Phobius, SignalP, SecretomeP, or TatP for extracellular secretion, and 11 contained transmembrane domain stretches suggested by Phobius and transmembrane hidden Markov model. The sample preparation technique demonstrating the most effective outcome for C. saccharolyticus secreted proteins in this study, involved acetone precipitation followed by the C₁₈ bead method in which 2.4% (63 proteins) of the predicted proteome was identified, including proteins suggested to have secretion and transmembrane moieties.     

Multiplexed optical coding nanobeads and their application in single-molecule counting analysis for multiple gene expression analysis

A method for fabrication of multiplexed optical coding nanobeads (MOCNBs) was developed by hybridizing three types of coding DNAs labeled with different dyes (Cy5, FAM and AMCA) at precisely controlled ratios with biotinylated reporter DNA modified to magnetic streptavidin-coated nanobeads with a diameter of 300 nm. The color of the MOCNBs could be observed by overlapping three single-primary-color fluorescence images of the MOCNBs corresponding to emission of Cy5 (red), FAM (green) and AMCA (blue). The MOCNBs could be easily identified under a conventional fluorescence microscope. The MOCNBs with different colors could serve as the multiplexed optical coding labels for single-molecule counting analysis (SMCA) and be used in multi-gene expression analysis (MGEA). In the SMCA-based MGEA technique, multiple messenger RNAs (mRNAs) in cells could be simultaneously quantified through their complementary DNAs (cDNAs) by counting the bright dots with the same color corresponding to the single cDNA molecules labeled with the MOCNBs. We measured expression profiles of three genes from Lepidoptera insect Helicoverpa armigera in ∼100 HaEpi cells with and without steroid hormone inductions to demonstrate the SMCA-based MGEA technique using MOCNBs.     

Comparison of stable-isotope labeling with amino acids in cell culture and spectral counting for relative quantification of protein expression

Protein quantification is one of the principal goals of mass spectrometry (MS)-based proteomics, and many strategies exist to achieve it. Several approaches involve the incorporation of a stable-isotope label using either chemical derivatization, enzymatically catalyzed incorporation of (18)O, or metabolic labeling in a cell or tissue culture. These techniques can be cost or time prohibitive or not amenable to the biological system of interest. Label-free techniques including those utilizing integrated ion abundance and spectral counting offer an alternative to stable-isotope-based methodologies. Herein, we present the comparison of stable-isotope labeling of amino acids in cell culture (SILAC) with spectral counting for the quantification of human embryonic stem cells as they differentiate toward the trophectoderm at three time points. Our spectral counting experimental strategy resulted in the identification of 2641 protein groups across three time points with an average sequence coverage of 30.3%, of which 1837 could be quantified with more than five spectral counts. SILAC quantification was able to identify 1369 protein groups with an average coverage of 24.7%, of which 1027 could be quantified across all time points. Within this context we further explore the capacity of each strategy for proteome coverage, variation in quantification, and the relative sensitivity of each technique to the detection of change in relative protein expression.     

Two-dimensional difference gel electrophoresis applied for analytical proteomics: fundamentals and applications to the study of plant proteomics

The present review reports the principles, fundamentals and some applications of two-dimensional difference gel electrophoresis for analytical proteomics based on plant proteome analysis, also emphasizing some advantages of 2-D DIGE over 2-D PAGE techniques. Some fluorescent protein labeling reagents, methods of protein labeling, models of 2-D DIGE experiments, and some limitations of this technique are presented and discussed in terms of 2-D DIGE plant proteomes. Finally, some practical applications of this technique are pointed out, emphasizing its potentialities in plant proteomics.     

Inverse 15N-metabolic labeling/mass spectrometry for comparative proteomics and rapid identification of protein markers/targets

The inverse labeling/mass spectrometry strategy has been applied to protein metabolic (15)N labeling for gel-free proteomics to achieve the rapid identification of protein markers/targets. Inverse labeling involves culturing both the perturbed (by disease or by a drug treatment) and control samples each in two separate pools of normal and (15)N-enriched culture media such that four pools are produced as opposed to two in a conventional labeling approach. The inverse labeling is then achieved by combining the normal (14)N-control with the (15)N-perturbed sample, and the (15)N-control with the (14)N-perturbed sample. Both mixtures are then proteolyzed and analyzed by mass spectrometry (coupled with on-line or off-line separation). Inverse labeling overcomes difficulties associated with protein metabolic labeling with regard to isotopic peak correlation and data interpretation in the single-experiment approach (due to the non-predictable/variable mass difference). When two data sets from inverse labeling are compared, proteins of differential expression are readily recognized by a characteristic inverse labeling pattern or apparent qualitative mass shifts between the two inverse labeling analyses. MS/MS fragmentation data provide further confirmation and are subsequently used to search protein databases for protein identification. The methodology has been applied successfully to two model systems in this study. Utilizing the inverse labeling strategy, one can use any mass spectrometer of standard unit resolution, and acquire only the minimum, essential data to achieve the rapid and unambiguous identification of differentially expressed protein markers/targets. The strategy permits quick focus on the signals of differentially expressed proteins. It eliminates the detection ambiguities caused by the dynamic range of detection. Finally, inverse labeling enables the detection of covalent changes of proteins responding to a perturbation that one might fail to distinguish with a conventional labeling experiment.     

Similarity between condensed phase and gas phase chemistry: Fragmentation of peptides containing oxidized cysteine residues and its implications for proteomics

Amino acid residues containing thioethers are easily oxidized during protein purification, derivatization, and/or digestion. For instance, oxidation of methionine residues in proteins during SDS-PAGE is commonly observed. Under low energy collision induced dissociation this gives rise to a second series of fragment ion of lower abundance that are shifted by -64 Da when compared to the oxidized methionine-containing fragments. We report here that alkylated cysteine residues can be found in their oxidized form too, indicating that the oxidation of thioethers can occur during and following protein digestion and not only during SDS-PAGE or reduction and alkylation. Collision induced dissociation experiments on the singly- and multiply-charged species reveals that these peptides preferentially undergo elimination reactions that forms a dehydroalanine from the oxidized, alkylated cysteine residue. This contrasts to the less abundant elimination reaction of peptides containing oxidized methionines which cannot form an alpha,beta-unsaturated compound, but parallels the condensed phased chemistry of sulfoxides. The masses of both precursor and product ions are shifted such that these peptides cannot be identified in database searches with current algorithms. Incorporation of this fragmentation pattern is important for the isotope-coded affinity tag approach since this method is based on peptides containing cysteine residues.