Deconvolution and Database Search of Complex Tandem Mass Spectra of Intact Proteins: A COMBINATORIAL APPROACH*

Top-down proteomics studies intact proteins, enabling new opportunities for analyzing post-translational modifications. Because tandem mass spectra of intact proteins are very complex, spectral deconvolution (grouping peaks into isotopomer envelopes) is a key initial stage for their interpretation. In such spectra, isotopomer envelopes of different protein fragments span overlapping regions on the m/z axis and even share spectral peaks. This raises both pattern recognition and combinatorial challenges for spectral deconvolution. We present MS-Deconv, a combinatorial algorithm for spectral deconvolution. The algorithm first generates a large set of candidate isotopomer envelopes for a spectrum, then represents the spectrum as a graph, and finally selects its highest scoring subset of envelopes as a heaviest path in the graph. In contrast with other approaches, the algorithm scores sets of envelopes rather than individual envelopes. We demonstrate that MS-Deconv improves on Thrash and Xtract in the number of correctly recovered monoisotopic masses and speed. We applied MS-Deconv to a large set of top-down spectra from Yersinia rohdei (with a still unsequenced genome) and further matched them against the protein database of related and sequenced bacterium Yersinia enterocolitica. MS-Deconv is available at http://proteomics.ucsd.edu/Software.html.Top-down proteomics is a mass spectrometry-based approach for identification of proteins and their post-translational modifications (PTMs)1 (1–14). Unlike the “bottom-up” approach where proteins are first digested into peptides and then a peptide mixture is analyzed by mass spectrometry, the top-down approach analyzes intact proteins. Thus, it has advantages in detecting and localizing PTMs as well as identifying multiple protein species (e.g. proteolytically processed protein species). Despite its advantages, top-down proteomics presents many challenges. These include requirement of high sample quantity, sophisticated instrumentation, protein separation, and robust computational analysis tools. For this reason, top-down proteomics has rarely been used for analyzing complex mixtures (12–18), and it is typically used to study single purified proteins. However, this situation is quickly changing with recent top-down studies of complex protein mixtures (14, 19).Because of the existence of natural isotopes, fragment ions of the same chemical formula and charge state are usually represented by a collection of spectral peaks in tandem mass spectra called an isotopomer envelope. The monoisotopic mass of a chemical formula is the sum of the masses of the atoms using the principal (most abundant) isotope for each element. Spectral deconvolution focuses on grouping spectral peaks into isotopomer envelopes. By doing so, the charge state and monoisotopic mass of each envelope are effectively determined. A complex multi-isotopic peak list in the m/z space is translated into a simple monoisotopic mass list that is easier to analyze.Given the monoisotopic mass and charge state of a fragment ion, its theoretical isotopic distribution can be predicted by assuming the fragment ion has an average elemental composition with respect to its mass (20) or using its precise elemental composition if the protein is known. Exploiting this, many deconvolution methods use theoretical isotopic distributions to detect and evaluate candidate isotopomer envelopes, which is the envelope detection problem (Fig. 1). To evaluate the fit of a candidate envelope to its theoretical isotopic distribution, many metrics have been proposed (20–32).Open in a separate windowFig. 1.Envelope detection. a, a theoretical isotopic distribution is predicted with the monoisotopic mass and charge state of a fragment ion. b, an observed envelope is detected by mapping peaks in the theoretical distribution to the spectrum. c, match between the theoretical isotopic distribution and the observed envelope. d, the theoretical isotopic distribution is scaled (the intensities of the peaks are multiplied by a constant) to have the best fit with the intensities of peaks in the observed envelope. Finally, a score for the observed envelope can be computed by comparing it with the intensity-scaled theoretical isotopic distribution.The candidate envelopes often overlap and share peaks, leading to a combinatorial problem of selecting the list of envelopes that best explains the spectrum (Fig. 2). In contrast to the well studied envelope detection problem, the envelope selection problem remains poorly explored. Most deconvolution algorithms follow a simple greedy approach to selecting the set of envelopes where the highest scoring envelopes are iteratively selected and removed from the spectrum. Although this approach often generates reasonable sets of envelopes for simple spectra, its performance deteriorates in cases of complex spectra.Open in a separate windowFig. 2.Envelope selection problem. Overlapping envelopes lead to a difficult combinatorial problem of selecting an optimal set of envelopes. We illustrate two cases where a deconvolution method that follows a greedy envelope selection outputs the envelope E₂, whereas the optimal solution consists of the envelopes E₁ and E₃. Example a illustrates the case where envelopes do not share peaks, and example b illustrates the case where envelopes share a spectral peak (E₁ and E₃).In particular, the greedy approach performs well when the envelopes are distributed sparsely along the m/z axis. Large proteins have many fragments that appear in multiple charge states. The high number of envelopes/peaks and the small m/z spread of the fragments with high charge states result in narrow m/z regions with high peak density. In these peak-dense regions, envelopes may overlap and share peaks, and the greedy approach and even manual interpretation often fail to find the optimal combination of envelopes (supplemental Fig. 1).Several methods have been proposed to explore the envelope selection problem. McIlwain et al. (33) presented a dynamic programming algorithm for selecting a set of envelopes such that the m/z ranges of the envelopes do not overlap. This non-overlapping condition becomes too restrictive for complex spectra of intact proteins. Samuelsson et al. (34) proposed a method that follows a non-negative sparse regression scheme. Du and Angeletti (35) and Renard et al. (36) addressed the envelope selection problem as a statistical problem of variable selection and used LASSO to solve it.Here, we present MS-Deconv, a combinatorial algorithm for spectral deconvolution. MS-Deconv (i) generates a large set of candidate envelopes, (ii) constructs an envelope graph encoding all envelopes and relationships between them, and (iii) finds a heaviest path in the envelope graph. Although the envelope graph of a complex spectrum is large (exceeding a million nodes in some cases), the heaviest path algorithm can efficiently find an optimal set of envelopes. MS-Deconv explicitly scores combinations of candidate envelopes rather than individual envelopes as in previous approaches.We tested MS-Deconv on a data set of top-down spectra from known proteins and evaluated the monoisotopic masses recovered by MS-Deconv. A mass was classified as a true positive if it was matched to the monoisotopic mass of a theoretical fragment ion of the protein within a specific parts per million (ppm) tolerance. We compared the performance of MS-Deconv with the widely used Thrash (20) and Xtract (37) and demonstrated that, with a few exceptions, MS-Deconv recovers more true positive masses. For example, for the collisionally activated dissociation (CAD) spectrum of bacteriorhodopsin (BR) with charge 10, the percentage of true positive masses among the top 150 masses is above 70% for MS-Deconv and less than 50% for Thrash. Additionally, MS-Deconv is ∼33 times faster than Thrash and 4 times faster than Xtract. Furthermore, MS-Deconv implements some user-friendly features: (i) outputs the set of peptide sequence tags, (ii) provides protein and spectral annotations, and (iii) allows one to inspect the recovered envelopes. We also tested MS-Deconv on a large LC-MS/MS data set from Yersinia rohdei (with a still unsequenced genome) (19). Y. rohdei is a non-pathogenic bacterium that is often used as a simulant for the potential bioterrorism agent Yersinia pestis, the causative agent of plague. We applied MS-Deconv to extract monoisotopic mass lists from top-down spectra and compared the mass lists with those reported by Thrash. We used ProSightPC (38) and the spectral alignment algorithm (39) to identify related proteins from a protein database of Yersinia enterocolitica (with a closely related and sequenced genome). The results demonstrated that MS-Deconv reported more matched fragments than Thrash for most proteins. Additionally, using spectral alignment, we identified eight proteins in Y. rohdei that were not reported in the ProSightPC-based searches (19) of the Y. enterocolitica protein database.     

Unrestrictive identification of post-translational modifications through peptide mass spectrometry

Proteins are post-translationally modified in vivo as part of cellular regulation and signaling, and undergo further chemical modifications during laboratory processing. Even relatively simple protein samples may carry a wide range of modifications. Peptide tandem mass spectrometry provides a way to study these events. We present a protocol for computational identification of post-translational modifications (PTMs) and the sites where they occur. The protocol performs an unrestrictive search, and requires no prior knowledge of what modifications are present in the sample. We present a largely automated procedure for PTM discovery, and provide a guide for analysis of PTM annotations. This protocol requires you to type out several commands, so you may wish to enlist the help of a colleague familiar with the computer's command-line interface. A typical MS run of up to 25,000 scans can be searched and analyzed in 3 h.     

Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility?

We have employed recently developed blind modification search techniques to generate the most comprehensive map of post-translational modifications (PTMs) in human lens constructed to date. Three aged lenses, two of which had moderate cataract, and one young control lens were analyzed using multidimensional liquid chromatography mass spectrometry. In total, 491 modification sites in lens proteins were identified. There were 155 in vivo PTM sites in crystallins: 77 previously reported sites and 78 newly detected PTM sites. Several of these sites had modifications previously undetected by mass spectrometry in lens including carboxymethyl lysine (+58 Da), carboxyethyl lysine (+72 Da), and an arginine modification of +55 Da with yet unknown chemical structure. These new modifications were observed in all three aged lenses but were not found in the young lens. Several new sites of cysteine methylation were identified indicating this modification is more extensive in lens than previously thought. The results were used to estimate the extent of modification at specific sites by spectral counting. We tested the long-standing hypothesis that PTMs contribute to age-related loss of crystallin solubility by comparing spectral counts between the water-soluble and water-insoluble fractions of the aged lenses and found that the extent of deamidation was significantly increased in the water-insoluble fractions. On the basis of spectral counting, the most abundant PTMs in aged lenses were deamidations and methylated cysteines with other PTMs present at lower levels.     

Anti-ulcer and antioxidant activity of Normacid, a herbomineral formulation

Effect of various doses (125, 250, 500 and 1000 mg/kg, po) of Normacid was studied on gastric secretion and gastric ulcers in pylorus-ligation and on ethanol-induced gastric mucosal injury in rats. The reduction in ulcer index in both the models along with the reduction in total acidity and an increase in the pH of gastric fluid in pylorus-ligated rats proved the anti-ulcer activity of Normacid. The increase in the levels of superoxide dismutase, catalase, reduced glutathione and membrane bound enzymes like Ca2+ ATPase, Mg2+ ATPase and Na+K+ ATPase and decrease in lipid peroxidation in both the models showed the antioxidant activity of the formulation. Thus it can be concluded that the anti-ulcer activity shown by Normacid may be due to the modulation of defensive factors by improvenent in gastric cytoprotection and partly due to antioxidant property.     

Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans

The egg jelly coats of sea urchins contain sulfated fucans which bind to a sperm surface receptor glycoprotein to initiate the signal transduction events resulting in the sperm acrosome reaction. The acrosome reaction is an ion channel regulated exocytosis which is an obligatory event for sperm binding to, and fusion with, the egg. Approximately 90% of individual females of the sea urchin Strongylocentrotus purpuratus spawned eggs having only one of two possible sulfated fucan electrophoretic isotypes, a slow migrating (sulfated fucan I), or a fast migrating (sulfated fucan II) isotype. The remaining 10% of females spawned eggs having both sulfated fucan isotypes. The two sulfated fucan isotypes were purified from egg jelly coats and their structures determined by NMR spectroscopy and methylation analysis. Both sulfated fucans are linear polysaccharides composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I is entirely sulfated at the O -2 position but with a heterogeneous sulfation pattern at O -4 position. Sulfated fucan II is composed of a regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p - 2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)- 1]n. Both purified sulfated fucans have approximately equal potency in inducing the sperm acrosome reaction. The significance of two structurally different sulfated fucans in the egg jelly coat of this species could relate to the finding that the sperm receptor protein which binds sulfated fucan contains two carbohydrate recognition modules of the C-type lectin variety which differ by 50% in their primary structure.      

Sequence variations in small-subunit ribosomal RNAs of Hartmannella vermiformis and their phylogenetic implications

Evidence of associations between free-living amoebas and human disease has been increasing in recent years. Knowledge about phylogenetic relationships that may be important for the understanding of pathogenicity in the genera involved is very limited at present. Consequently, we have begun to study these relationships and report here on the phylogeny of Hartmannella vermiformis, a free-living amoeba that can harbor the etiologic agent of Legionnaires' disease. Our analysis is based on studies of small-subunit ribosomal RNA genes (srDNA). Nucleotide sequences were determined for nuclear srDNA from three strains of H. vermiformis isolated from the United Kingdom, Germany, and the United States. These sequences then were compared with a sequence previously obtained for a North American isolate by J. H. Gunderson and M. L. Sogin. The four genes are 1,840 bp long, with an average GC content of 49.6%. Sequence differences among the strains range are 0.38%-0.76%. Variation occurs at 19 positions and includes 2 single-base indels plus 14 monotypic and 3 ditypic single-base substitutions. Variation is limited to eight helix/loop structures according to a current model for srRNA secondary structure. Parsimony, distance, and bootstrap analyses used to examine phylogenetic relationships between the srDNA sequences of H. vermiformis and other eukaryotes indicated that Hartmannella sequences were most closely related to those of Acanthamoeba and the alga Cryptomonas. All ditypic sites were consistent with a separation between European and North American strains of Hartmannella, but results of other tests of this relationship were statistically inconclusive.      

Cloning and characterization of SK2 channel from chicken short hair cells

In the inner ear of birds, as in mammals, reptiles and amphibians, acetylcholine released from efferent neurons inhibits hair cells via activation of an apamin-sensitive, calcium-dependent potassium current. The particular potassium channel involved in avian hair cell inhibition is unknown. In this study, we cloned a small-conductance, calcium-sensitive potassium channel (gSK2) from a chicken cochlear library. Using RT-PCR, we demonstrated the presence of gSK2 mRNA in cochlear hair cells. Electrophysiological studies on transfected HEK293 cells showed that gSK2 channels have a conductance of approximately 16 pS and a half-maximal calcium activation concentration of 0.74±0.17 M. The expressed channels were blocked by apamin (IC₅₀=73.3±5.0 pM) and d-tubocurarine (IC₅₀=7.6±1.0 M), but were insensitive to charybdotoxin. These characteristics are consistent with those reported for acetylcholine-induced potassium currents of isolated chicken hair cells, suggesting that gSK2 is involved in efferent inhibition of chicken inner ear. These findings imply that the molecular mechanisms of inhibition are conserved in hair cells of all vertebrates.     

The number of recombination events in a sample history: conflict graph and lower bounds

We consider the following problem: Given a set of binary sequences, determine lower bounds on the minimum number of recombinations required to explain the history of the sample, under the infinite-sites model of mutation. The problem has implications for finding recombination hotspots and for the Ancestral Recombination Graph reconstruction problem. Hudson and Kaplan gave a lower bound based on the four-gamete test. In practice, their bound R/sub m/ often greatly underestimates the minimum number of recombinations. The problem was recently revisited by Myers and Griffiths, who introduced two new lower bounds R/sub h/ and R/sub s/ which are provably better, and also yield good bounds in practice. However, the worst-case complexities of their procedures for computing R/sub h/ and R/sub s/ are exponential and super-exponential, respectively. In this paper, we show that the number of nontrivial connected components, R/sub c/, in the conflict graph for a given set of sequences, computable in time 0(nm/sup 2/), is also a lower bound on the minimum number of recombination events. We show that in many cases, R/sub c/ is a better bound than R/sub h/. The conflict graph was used by Gusfield et al. to obtain a polynomial time algorithm for the galled tree problem, which is a special case of the Ancestral Recombination Graph (ARG) reconstruction problem. Our results also offer some insight into the structural properties of this graph and are of interest for the general Ancestral Recombination Graph reconstruction problem.     

SCOPE: a probabilistic model for scoring tandem mass spectra against a peptide database

Proteomics, or the direct analysis of the expressed protein components of a cell, is critical to our understanding of cellular biological processes in normal and diseased tissue. A key requirement for its success is the ability to identify proteins in complex mixtures. Recent technological advances in tandem mass spectrometry has made it the method of choice for high-throughput identification of proteins. Unfortunately, the software for unambiguously identifying peptide sequences has not kept pace with the recent hardware improvements in mass spectrometry instruments. Critical for reliable high-throughput protein identification, scoring functions evaluate the quality of a match between experimental spectra and a database peptide. Current scoring function technology relies heavily on ad-hoc parameterization and manual curation by experienced mass spectrometrists. In this work, we propose a two-stage stochastic model for the observed MS/MS spectrum, given a peptide. Our model explicitly incorporates fragment ion probabilities, noisy spectra, and instrument measurement error. We describe how to compute this probability based score efficiently, using a dynamic programming technique. A prototype implementation demonstrates the effectiveness of the model.