Rapid Validation of Mascot Search Results via Stable Isotope Labeling, Pair Picking, and Deconvolution of Fragmentation Patterns

Conventional LC-MS/MS data analysis matches each precursor ion and fragmentation pattern to their best fit within databases of theoretical spectra, yielding a peptide identification. Confidence is estimated by a score but can be validated by statistics, false discovery rates, and/or manual validation. A weakness is that each ion is evaluated independently, discarding potentially useful cross-correlations. In a classical approach to de novo sequence analysis, mixtures of peptides differing only in a carboxyl-terminal isotopic label yield fragmentation spectra with single, unlabeled b-type ions but pairs of isotope-labeled y-type ions, facilitating confident assignments. To apply this principle to identification by fragmentation pattern matching, we developed Validator, software that recognizes isotopic peptide pairs and compares their identifications and fragmentation patterns. Testing Validator 1 on a Mascot results file from FT-ICR LC-MS/MS of ¹⁶O/¹⁸O-labeled yeast cell lysate peptides yielded 2,775 peptide pairs sharing a common identification but differing in carboxyl-terminal label. Comparing observed b- and y-ions with the predicted fragmentation pattern improved the threshold Mascot score for 5% false discovery from 36 to 22, significantly increasing both sensitivity and specificity. Validator 2, which identifies pairs by precursor mass difference alone before comparing observed fragmentation with that predicted by Mascot, found 2,021 isotopic pairs, similarly achieving improved sensitivity and specificity. Finally Validator 3, which finds pairs based on mass difference alone and then deconvolutes fragmentation patterns independently of Mascot, found 964 predicted peptides. Validator 3 allowed raw mass spectrometry data to be mined not only to validate Mascot results but also to discover peptides missed by Mascot. Using standard desktop hardware, the Validator 1–3 software processed the 11,536 spectra in the 93-MB Mascot .DAT file in less than 6 min (32 spectra/s), revealing high confidence peptide identifications without regard to Mascot score, far faster than manual or other independent validation methods.MS/MS combined with informatics analysis is now a uniquely powerful approach for identifying the components of complex protein samples (1–3). Although new technologies have dramatically enhanced the speed, sensitivity, and precision of LC-MS/MS instrumentation (4), data analysis has neither kept pace with nor taken full advantage of these advances. Determining peptide sequences from fragment ion spectra remains a difficult problem, and three main strategies have matured (5). In de novo sequencing, the peptide sequence is inferred directly from the fragment ion spectra, and many algorithms have been developed to automate this process, including Lutefisk (6), PepNovo (7), NovoHMM (8), Peptide Identification via Integer linear Optimization (PILOT) (9), and others (10–13). Incomplete fragmentation patterns and low signal to noise (10) make this method difficult to implement as an exclusive means of peptide identification.The most commonly used method involves comparing experimental MS/MS spectra to theoretical peptide fragmentation patterns derived from protein sequence databases (4) and reporting the best peptide match, which is then propagated forward through the process of determining likely protein components. Several programs are commonly used, including SEQUEST (14, 15), Mascot (16), and X! Tandem (17, 18). What these algorithms share is the determination of a score for a spectrum-peptide match and subsequently a protein identification, and it is the way in which these scores are assigned and interpreted that distinguishes them (19).The third method for spectrum-peptide matching is a hybrid of de novo and database searching (5) in which small lengths of sequence are generated directly from the fragment ion spectra, and these “sequence tags” (20) are used to corroborate spectrum-database matches. Popular implementations of this strategy include DirecTag (21), GutenTag (22), and MultiTag (23). The limitations to this method include the requirement for consecutive fragmentation ions and the reliance on de novo algorithms to identify sequence tags.Database search is highly susceptible to both overreporting false positives (low specificity) and underreporting true positives (low sensitivity). The search engines provide different scoring systems that cannot be directly compared, as the rankings of spectral quality are often based on arbitrary cutoff values. Recent research has focused less on the sequence matching algorithms themselves but more on the statistics used to evaluate the resulting match scores (24). PeptideProphet was one of the first algorithms developed to evaluate match scores and assign probabilities by evaluating each match with respect to all other peptide assignments. By using machine learning techniques (an expectation-maximization algorithm), PeptideProphet was shown to have high discriminating power for database search results (25). Initially developed for SEQUEST search results, PeptideProphet has been subsequently adapted for use with database search results from Mascot and X! Tandem. These components are combined in Scaffold, a commercial software suite developed by Proteome Software. An alternative approach is to filter the primary data to exclude poor quality MS/MS scans prior to the database search (26), thereby enhancing the likely significance of each reported match.Using a false discovery rate instead of a false-positive rate is now the standard statistical measure for reporting error rates in data sets with large numbers of features (e.g. proteomics or genomics data) (5, 27). Target-decoy searching as an estimate of false discovery rate (FDR)¹ involves first constructing a database of decoy peptides (28, 29), and this strategy is being incorporated into PeptideProphet (30, 31). For each peptide-spectrum match, the target spectrum is queried against a second (decoy) database with characteristics similar to those of the first (e.g. a database of reversed or random peptides). Matches to the decoy database are considered false discoveries, and the number of matches above a particular cutoff score threshold is reported. The target-decoy search option is now available in the newest version (version 2.2) of the database search engine Mascot (Matrix Science).Despite these advances in mass spectrometry, database searching, and statistical approaches to validating matches, the process of analyzing mass spectrometry data remains time-consuming and computer processor-intensive, often requiring several steps and various data transformations (19). To overcome these limitations, we developed a fast and efficient method for peptide identification validation that minimizes the false discovery rate. Our algorithm relies on data from stable isotopic labeling, which is a standard method for quantifying relative protein abundance in complex mixtures (see Ref. 32 and references therein). Carboxyl-terminal labeling methods, including trypsin-catalyzed ¹⁸O exchange (33), result in a mixture of pairs of chemically identical but isotopically distinct peptides. The “light” and “heavy” peptides co-elute from HPLC but are readily distinguished by precursor mass (Fig. 1A). Each peptide also has an isotopic envelope comprised of isotopologues, molecules that are identical in composition except they can contain any number of isotopes. In the case of trypsin-catalyzed ¹⁸O exchange, two ¹⁸O atoms are substituted for the two carboxyl-terminal ¹⁶O atoms. Comparison of CID fragmentation patterns of carboxyl terminus-labeled light and heavy precursors (or isotopologues) distinguishes b-type and y-type ions (34, 35). The carboxyl-terminal fragments (y-ions) appear as light (¹⁶O) and heavy (¹⁸O-substituted) forms, but the amino-terminal fragments (b-ions) display a single shared mass (Fig. 1, B–D).Open in a separate windowFig. 1.Peptide pair identification strategy. A, shown is an example of experimental spectra of a ¹⁶O/¹⁸O-peptide pair. Each peptide has an isotopic envelope comprised of three to four different isotopologues containing zero to three molecules of ¹³C, ¹⁵N, or other naturally occurring stable isotopes. The ¹⁸O envelope is shifted by about 2.0 Da, reflecting the difference in mass due to the substitution of two ¹⁸O atoms. Note that the difference of 2.0 Da is due to the peptide having a 2+ charge state. Peptide pairs with a 1+ charge would be separated by about 4.0 Da. B, the b-type and y-type ions from the collision-induced dissociation of a peptide are shown. Any carboxyl-terminal substitution (as in ¹⁸O, indicated by *) will affect the y-ions exclusively. C, idealized sample MS/MS spectra from the peptide and ions in B. The spectra from the ¹⁶O- and ¹⁸O-peptide forms have similar patterns, although the peak heights may be different. D, top, the two spectra from C are overlaid to demonstrate that the b-ions will have a nearly identical mass-to-charge ratio, whereas the y-ions will have a shift reflective of the stable isotope substitution. In the example given, peaks “a” and “k” from C are both b-ions and therefore overlap, whereas peaks “b” and “l” are y-ions with l being shifted due to the substitution of two ¹⁸O atoms. Shifted ions are indicated with a horizontal bar underneath. By observing which ions overlap and which have shifted, the identities of the b- and y-ions can be inferred (D, bottom).The technique of using isotopic pairs to enhance peptide identification is not new, and several authors have recognized that isotopic labeling could be used to differentiate carboxyl-terminal from amino-terminal peptide fragments to facilitate peptide sequence analysis (2, 33, 35–38). This method has been productively applied to de novo analysis (12, 39–45) and peptide mass fingerprinting (46). In addition, analogous techniques have been applied to the analysis of mixtures of modified and unmodified peptides by probing for peptide mass differences that match known post-translational modifications (47); other groups have used MS/MS spectra information to corroborate these matches and remove noise (48, 49). Finally, isotopic labeling with ¹⁸O has been used for manual validation of peptide identifications by observing the predicted mass shift of y-ions (50). Nevertheless, this strategy has yet to be harnessed as a means for automated data analysis and peptide search validation.The goal of this study was to develop a set of software tools designed to provide rapid and automatic validation of peptide assignments by Mascot and to determine the relative benefit of reducing false discovery and the magnitude of loss of bona fide identifications. We hypothesized that the characteristic shifting of y-type ions between fragmentation spectra of light and heavy precursors might provide a robust check for validity of peptide assignment by database search. Here we demonstrate the feasibility of quickly and efficiently analyzing searched mass spectrometry data, determining within minutes which peptide and protein assignments are likely valid. In its simplest form, Validator 1, identified isotopic pairs in a Mascot results file and improved the 5% FDR cutoff from a Mascot score of 36 to 22, thereby capturing many true identifications that would otherwise have been discarded. A more advanced algorithm, Validator 3, that considers only precursor ion mass, charge, and fragmentation spectral data to identify isotopic pairs independently of any peptide identifications, not only rapidly validated the Mascot results but also discovered peptides that Mascot had failed to match. Our software suite, Validator 1–3, provides new and robust tools for rapid validation of searched LC-MS/MS data obtained in stable isotope experiments, offering improved sensitivity and specificity over database searching alone.     

Alveolar macrophage activation is a key initiation signal for acute lung ischemia-reperfusion injury

Lung ischemia-reperfusion (I/R) injury is a biphasic inflammatory process. Previous studies indicate that the later phase is neutrophil-dependent and that alveolar macrophages (AMs) likely contribute to the acute phase of lung I/R injury. However, the mechanism is unclear. AMs become activated and produce various cytokines and chemokines in many inflammatory responses, including transplantation. We hypothesize that AMs respond to I/R by producing key cytokines and chemokines and that depletion of AMs would reduce cytokine/chemokine expression and lung injury after I/R. To test this, using a buffer-perfused, isolated mouse lung model, we studied the impact of AM depletion by liposome-clodronate on I/R-induced lung dysfunction/injury and expression of cytokines/chemokines. I/R caused a significant increase in pulmonary artery pressure, wet-to-dry weight ratio, vascular permeability, tumor necrosis factor (TNF)-alpha, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-2 expression, as well as decreased pulmonary compliance, when compared with sham lungs. After AM depletion, the changes in each of these parameters between I/R and sham groups were significantly attenuated. Thus AM depletion protects the lungs from I/R-induced dysfunction and injury and significantly reduces cytokine/chemokine production. Protein expression of TNF-alpha and MCP-1 are positively correlated to I/R-induced lung injury, and AMs are a major producer/initiator of TNF-alpha, MCP-1, and MIP-2. We conclude that AMs are an essential player in the initiation of acute lung I/R injury.     

A novel mechanism of ion homeostasis and salt tolerance in yeast: the Hal4 and Hal5 protein kinases modulate the Trk1-Trk2 potassium transporter

The regulation of intracellular ion concentrations is a fundamental property of living cells. Although many ion transporters have been identified, the systems that modulate their activity remain largely unknown. We have characterized two partially redundant genes from Saccharomyces cerevisiae, HAL4/SAT4 and HAL5, that encode homologous protein kinases implicated in the regulation of cation uptake. Overexpression of these genes increases the tolerance of yeast cells to sodium and lithium, whereas gene disruptions result in greater cation sensitivity. These phenotypic effects of the mutations correlate with changes in cation uptake and are dependent on a functional Trk1-Trk2 potassium transport system. In addition, hal4 hal5 and trk1 trk2 mutants exhibit similar phenotypes: (i) they are deficient in potassium uptake; (ii) their growth is sensitive to a variety of toxic cations, including lithium, sodium, calcium, tetramethylammonium, hygromycin B, and low pH; and (iii) they exhibit increased uptake of methylammonium, an indicator of membrane potential. These results suggest that the Hal4 and Hal5 protein kinases activate the Trk1-Trk2 potassium transporter, increasing the influx of potassium and decreasing the membrane potential. The resulting loss in electrical driving force reduces the uptake of toxic cations and improves salt tolerance. Our data support a role for regulation of membrane potential in adaptation to salt stress that is mediated by the Hal4 and Hal5 kinases.     

Can Dreams During Pregnancy Predict Postpartum Depression?

Postpartum depression (henceforth PPD) is an emotional disturbance which occurs in as much as 20% of the childbearing population. This study attempted to ascertain whether dreams, offering unconscious expression of internal emotional processes, could help to identify early signs of PPD. It was hypothesized that differences would be found in the emotional dream work of pregnant women who either later developed or did not develop PPD. 166 women participated in the two stages of the study. During stage I, the women were interviewed in the last trimester of their first pregnancies. The interview included a demographic questionnaire and an account of a dream. During stage II, the women were interviewed 6–10 weeks after giving birth. The second interview included only the EPDS scale for affirming or denying the occurrence of PPD. The findings of the study confirm the hypothesis that dreams of pregnant women can differentiate among women who are or are not at-risk for PPD. It was found that more unpleasant dreams and dreams expressing apprehension were found among women who did not later develop PPD, than among women who did develop PPD.     

Yeast cell death during DNA damage arrest is independent of caspase or reactive oxygen species

CDC13 encodes a telomere-binding protein that prevents degradation of telomeres. cdc13-1 yeast grown at the nonpermissive temperature undergo G2/M arrest, progressive chromosome instability, and subsequent cell death. Recently, it has been suggested that cell death in the cdc13-1 mutant is an active process characterized by phenotypic hallmarks of apoptosis and caspase activation. In this work, we show that cell death triggered by cdc13-1 is independent of the yeast metacaspase Yca1p and reactive oxygen species but related to cell cycle arrest per se. Inactivating YCA1 or depleting reactive oxygen species does not increase viability of cdc13-1 cells. In turn, caspase activation does not precede cell death in the cdc13-1 mutant. Yca1p activity assayed by cell binding of mammalian caspase inhibitors is confounded by artifactual labeling of dead yeast cells, which nonspecifically bind fluorochromes. We speculate that during a prolonged cell cycle arrest, cdc13-1 cells reach a critical size and die by cell lysis.     

Assaying Bcr-Abl kinase activity and inhibition in whole cell extracts by phosphorylation of substrates immobilized on agarose beads

There is a current and increasing demand for simple, robust, nonradioactive assays of protein tyrosine kinase activity with applications for clinical diagnosis and high-throughput screening of potential molecularly targeted therapeutic agents. One significant challenge is to detect and measure the activity of specific kinases with key roles in cell signaling as an approach to distinguish normal cells from cancer cells and as a means of evaluating targeted drug efficacy and resistance in cancer cells. Here, we describe a method in which kinase substrates fused to glutathione-S-transferase and immobilized on glutathione agarose beads are phosphorylated, eluted, and then assayed to detect kinase activity. The activity of recombinant, purified c-Abl kinase or Bcr-Abl kinase in whole cell extracts can be detected with equivalent specificity, sensitivity, and reproducibility. Similarly, inhibition of recombinant c-Abl or Bcr-Abl in cells or cell extracts by imatinib mesylate and other Bcr-Abl targeted kinase inhibitors is readily assayed. This simple kinase assay is sufficiently straightforward and robust for use in clinical laboratories and is potentially adaptable to high-throughput assay formats.     

Functional dissection of a conserved motif within the pilus retraction protein PilT

PilT is a hexameric ATPase required for type IV pilus retraction in gram-negative bacteria. Retraction of type IV pili mediates intimate attachment to and signaling in host cells, surface motility, biofilm formation, natural transformation, and phage sensitivity. We investigated the in vivo and in vitro roles of each amino acid of the distinct, highly conserved C-terminal AIRNLIRE motif in PilT. Substitution of amino acids A288, I289, L292, and I293 as well as a double substitution of R290 and R294 abolished Pseudomonas aeruginosa PilT function in vivo, as measured by a loss of surface motility and phage sensitivity. When introduced into purified Aquifex aeolicus PilT, substitutions in the AIRNLIRE motif did not disrupt ATPase activity or oligomerization. In contrast, a K136Q substitution in the broadly conserved nucleotide binding motif prevented PilT function in vivo as well as in vitro. We propose that the AIRNLIRE motif forms an amphipathic alpha helix which transmits signals between a surface-exposed protein interaction site and the ATPase core of PilT, and we recognize a potential functional homology in other type II secretion ATPases.     

Human anti-asparaginyl-tRNA synthetase autoantibodies (anti-KS) increase the affinity of the enzyme for its tRNA substrate

Autoantibodies directed against specific human aminoacyl-tRNA synthetases have been associated with a clinical picture including myositis, arthritis, interstitial lung disease and other features that has been referred to as the "anti-synthetase syndrome". Anti-asparaginyl-tRNA synthetase autoantibodies (anti-KS), the most recently described anti-synthetase autoantibodies, are directed against human cytosolic asparaginyl-tRNA synthetase and neutralize specifically its activity. Here we show that these antibodies recognize two epitopes on the human enzyme, an N-terminal epitope reactive in immunoblot experiments and a heat-labile epitope in the catalytic domain. In contrast to the well studied anti-Jo-1 autoantibodies anti-KS when bound to the synthetase increase the affinity of the synthetase for its tRNA substrate and prevent aminoacylation without interfering with the amino acid activation step.     

Yng2p-dependent NuA4 histone H4 acetylation activity is required for mitotic and meiotic progression.

In all eukaryotes, multisubunit histone acetyltransferase (HAT) complexes acetylate the highly conserved lysine residues in the amino-terminal tails of core histones to regulate chromatin structure and gene expression. One such complex in yeast, NuA4, specifically acetylates nucleosome-associated histone H4. Recent studies have revealed that NuA4 comprises at least 11 subunits, including Yng2p, a yeast homolog of the candidate human tumor suppressor gene, ING1. Consistent with prior data, we find that cells lacking Yng2p are deficient for NuA4 activity and are temperature-sensitive. Furthermore, we show that the NuA4 complex is present in the absence of Yng2p, suggesting that Yng2p functions to maintain or activate NuA4 HAT activity. Sporulation of diploid yng2 mutant cells reveals a defect in meiotic progression, whereas synchronized yng2 mutant cells display a mitotic delay. Surprisingly, genome-wide expression analysis revealed little change from wild type. Nocodazole arrest and release relieves the mitotic defects, suggesting that Yng2p may have a critical function prior to or during metaphase. Rather than a uniform decrease in acetylated forms of histone H4, we find striking cell-to-cell heterogeneity in the loss of acetylated histone H4 in yng2 mutant cells. Treating yng2 mutants with the histone deacetylase inhibitor trichostatin A suppressed the mitotic delay and restored global histone H4 acetylation, arguing that reduced H4 acetylation may underlie the cell cycle delay.