Tactical Voting in Plurality Elections

How often will elections end in landslides? What is the probability for a head-to-head race? Analyzing ballot results from several large countries rather anomalous and yet unexplained distributions have been observed. We identify tactical voting as the driving ingredient for the anomalies and introduce a model to study its effect on plurality elections, characterized by the relative strength of the feedback from polls and the pairwise interaction between individuals in the society. With this model it becomes possible to explain the polarization of votes between two candidates, understand the small margin of victories frequently observed for different elections, and analyze the polls'' impact in American, Canadian, and Brazilian ballots. Moreover, the model reproduces, quantitatively, the distribution of votes obtained in the Brazilian mayor elections with two, three, and four candidates.     

Ecosystem-level effects of re-oligotrophication and N:P imbalances in rivers and estuaries on a global scale

Trends and ecological consequences of phosphorus (P) decline and increasing nitrogen (N) to phosphorus (N:P) ratios in rivers and estuaries are reviewed and discussed. Results suggest that re-oligotrophication is a dominant trend in rivers and estuaries of high-income countries in the last two–three decades, while in low-income countries widespread eutrophication occurs. The decline in P is well documented in hundreds of rivers of United States and the European Union, but the biotic response of rivers and estuaries besides phytoplankton decline such as trends in phytoplankton composition, changes in primary production, ecosystem shifts, cascading effects, changes in ecosystem metabolism, etc., have not been sufficiently monitored and investigated, neither the effects of N:P imbalance. N:P imbalance has significant ecological effects that need to be further investigated. There is a growing number of cases in which phytoplankton biomass have been shown to decrease due to re-oligotrophication, but the potential regime shift from phytoplankton to macrophyte dominance described in shallow lakes has been documented only in a few rivers and estuaries yet. The main reasons why regime shifts are rarely described in rivers and estuaries are, from one hand the scarcity of data on macrophyte cover trends, and from the other hand physical factors such as peak flows or high turbidity that could prevent a general spread of submerged macrophytes as observed in shallow lakes. Moreover, re-oligotrophication effects on rivers may be different compared to lakes (e.g., lower dominance of macrophytes) or estuaries (e.g., limitation of primary production by N instead of P) or may be dependent on river/estuary type. We conclude that river and estuary re-oligotrophication effects are complex, diverse and still little known, and in some cases are equivalent to those described in shallow lakes, but the regime shift is more likely to occur in mid to high-order rivers and shallow estuaries.     

The detection and attribution of extreme reductions in vegetation growth across the global land surface

Negative extreme anomalies in vegetation growth (NEGs) usually indicate severely impaired ecosystem services. These NEGs can result from diverse natural and anthropogenic causes, especially climate extremes (CEs). However, the relationship between NEGs and many types of CEs remains largely unknown at regional and global scales. Here, with satellite-derived vegetation index data and supporting tree-ring chronologies, we identify periods of NEGs from 1981 to 2015 across the global land surface. We find 70% of these NEGs are attributable to five types of CEs and their combinations, with compound CEs generally more detrimental than individual ones. More importantly, we find that dominant CEs for NEGs vary by biome and region. Specifically, cold and/or wet extremes dominate NEGs in temperate mountains and high latitudes, whereas soil drought and related compound extremes are primarily responsible for NEGs in wet tropical, arid and semi-arid regions. Key characteristics (e.g., the frequency, intensity and duration of CEs, and the vulnerability of vegetation) that determine the dominance of CEs are also region- and biome-dependent. For example, in the wet tropics, dominant individual CEs have both higher intensity and longer duration than non-dominant ones. However, in the dry tropics and some temperate regions, a longer CE duration is more important than higher intensity. Our work provides the first global accounting of the attribution of NEGs to diverse climatic extremes. Our analysis has important implications for developing climate-specific disaster prevention and mitigation plans among different regions of the globe in a changing climate.     

Synthesis of 2-ethylhexyl oleate catalyzed by Candida antarctica lipase immobilized on a magnetic polymer support in continuous flow

Bioprocess and Biosystems Engineering - This study investigated the synthesis of 2-ethylhexyl oleate catalyzed by Candida antarctica lipase immobilized on magnetic poly(styrene-co-divinylbenzene)...     

Role of AIP56 disulphide bond and its reduction by cytosolic redox systems for efficient intoxication

Apoptosis‐inducing protein of 56 kDa (AIP56) is a major virulence factor of Photobacterium damselae subsp. piscicida, a gram‐negative pathogen that infects warm water fish species worldwide and causes serious economic losses in aquacultures. AIP56 is a single‐chain AB toxin composed by two domains connected by an unstructured linker peptide flanked by two cysteine residues that form a disulphide bond. The A domain comprises a zinc‐metalloprotease moiety that cleaves the NF‐kB p65, and the B domain is involved in binding and internalisation of the toxin into susceptible cells. Previous experiments suggested that disruption of AIP56 disulphide bond partially compromised toxicity, but conclusive evidences supporting the importance of that bond in intoxication were lacking. Here, we show that although the disulphide bond of AIP56 is dispensable for receptor recognition, endocytosis, and membrane interaction, it needs to be intact for efficient translocation of the toxin into the cytosol. We also show that the host cell thioredoxin reductase‐thioredoxin system is involved in AIP56 intoxication by reducing the disulphide bond of the toxin at the cytosol. The present study contributes to a better understanding of the molecular mechanisms operating during AIP56 intoxication and reveals common features shared with other AB toxins.     

The relative importance of spatial and environmental processes in the assembly of larval Chironomidae (Insecta,Diptera) communities along a transition landscape in southern Brazilian streams

Limnology - Metacommunity structure of stream invertebrates is contingent on complex interplays between species dispersal ability, spatial extent and watershed environmental specificities. Previous...     

Phylogeography and genetic diversity of the commercially-collected Caribbean blue-legged hermit crab (Clibanarius tricolor)

Shifts from ornamental fish tanks to functional reef ecosystem aquaria has led to unprecedented pressure on marine invertebrate fisheries. The Caribbean blue-legged hermit crab (Clibanarius tricolor) is among the species targeted for functional reef tanks, valued for its role as an aquarium cleaner. Little is known about the biology of the species or the genetic landscape in which the increased collecting is happening. Here, we investigate the phylogeographic history and genetic diversity of C. tricolor through analysis of mitochondrial (CO1, 16S) and nuclear (H3) DNA. We test whether phylogeographic breaks for other invertebrates structure the genetic diversity of C. tricolor and explore additional factors that may govern structure, such as reproductive strategy, life history, habitat preference, adult mobility, demographic history, and vicariance events. Based on these three markers, we find high genetic diversity and connectivity and find no evidence to support the tested barriers as relevant to gene flow for C. tricolor. Rather, mitochondrial and nuclear markers infer high genetic diversity, panmixia, and demographic expansion during the Pleistocene. Our finding of panmixia makes it difficult to identify source or sink populations, but the absence of hierarchical structure inferred from mtDNA and nuDNA markers we use, high levels of genetic diversity and homogeneity for these same markers, and advantageous life history traits suggest C. tricolor is not currently at special risk; however, geographically restricted haplotypes and limitations within our study prevent us from making a strong conclusion about the sustainability of the fishery. Our work on the Caribbean blue-legged hermit crab highlights the importance of acquiring basic information on exploited species and reiterates that common regional forces may not equally impact connectivity among co-distributed species.

     

P-selectin glycoprotein ligand 1 promotes T cell lymphoma development and dissemination

P-selectin glycoprotein ligand-1 (PSGL-1) is a membrane-bound glycoprotein expressed in lymphoid and myeloid cells. It is a ligand of P-, E- and L-selectin and is involved in T cell trafficking and homing to lymphoid tissues, among other functions. PSGL-1 expression has been implicated in different lymphoid malignancies, so here we aimed to evaluate the involvement of PSGL-1 in T cell lymphomagenesis and dissemination. PSGL-1 was highly expressed at the surface of human and mouse T cell leukemia and lymphoma cell lines. To assess its impact on T cell malignancies, we stably expressed human PSGL-1 (hPSGL-1) in a mouse thymic lymphoma cell line, which expresses low levels of endogenous PSGL-1 at the cell surface. hPSGL-1-expressing lymphoma cells developed subcutaneous tumors in athymic nude mice recipients faster than control empty vector or parental cells. Moreover, the kidneys, lungs and liver of tumor-bearing mice were infiltrated by hPSGL-1-expressing malignant T cells. To evaluate the role of PSGL-1 in lymphoma cell dissemination, we injected intravenously control and hPSGL-1-expressing lymphoma cells in athymic mice. Strikingly, PSGL-1 expression facilitated disease infiltration of the kidneys, as determined by histological analysis and anti-CD3 immunohistochemistry. Together, these results indicate that PSGL-1 expression promotes T cell lymphoma development and dissemination to different organs.     

Determination of protein concentration in downstream biomanufacturing processes by in-line index of refraction

Protein concentration determination is a necessary in-process control for the downstream operations within biomanufacturing. As production transitions from batch mode to an integrated continuous bioprocess paradigm, there is a growing need to move protein concentration quantitation from off-line to in-line analysis. One solution to fulfill this process analytical technology need is an in-line index of refraction (IoR) sensor to measure protein concentration in real time. Here the performance of an IoR sensor is evaluated through a series of experiments to assess linear response, buffer matrix effects, dynamic range, sensor-to-sensor variability, and the limits of detection and quantitation. The performance of the sensor was also tested in two bioprocessing scenarios, ultrafiltration and capture chromatography. The implementation of this in-line IoR sensor for real-time protein concentration analysis and monitoring has the potential to improve continuous bioprocess manufacturing.     

Neutron-encoded Signatures Enable Product Ion Annotation From Tandem Mass Spectra

We report the use of neutron-encoded (NeuCode) stable isotope labeling of amino acids in cell culture for the purpose of C-terminal product ion annotation. Two NeuCode labeling isotopologues of lysine, ¹³C₆¹⁵N₂ and ²H₈, which differ by 36 mDa, were metabolically embedded in a sample proteome, and the resultant labeled proteins were combined, digested, and analyzed via liquid chromatography and mass spectrometry. With MS/MS scan resolving powers of ∼50,000 or higher, product ions containing the C terminus (i.e. lysine) appear as a doublet spaced by exactly 36 mDa, whereas N-terminal fragments exist as a single m/z peak. Through theory and experiment, we demonstrate that over 90% of all y-type product ions have detectable doublets. We report on an algorithm that can extract these neutron signatures with high sensitivity and specificity. In other words, of 15,503 y-type product ion peaks, the y-type ion identification algorithm correctly identified 14,552 (93.2%) based on detection of the NeuCode doublet; 6.8% were misclassified (i.e. other ion types that were assigned as y-type products). Searching NeuCode labeled yeast with PepNovo⁺ resulted in a 34% increase in correct de novo identifications relative to searching through MS/MS only. We use this tool to simplify spectra prior to database searching, to sort unmatched tandem mass spectra for spectral richness, for correlation of co-fragmented ions to their parent precursor, and for de novo sequence identification.The ability to make de novo sequence identifications directly from tandem mass spectra has long been a holy grail of the proteomic community. Such a capability would wean the field from its reliance upon sequenced genome databases. Even for organisms with fully annotated genomes, events such as single nucleotide polymorphisms, alternative splicing, gene fusion, and a host of other genomic transformations can result in altered proteomes. These alterations can vary from cell to cell and individual to individual. Thus, one could argue that the most valuable proteomic information, the individual and cellular proteome variation from the genome, remains elusive (1). This problem has received considerable attention; that said, it is not easy to de novo correlate spectrum to sequence in a large-scale, automated fashion (2–6). Improvements in mass accuracy have helped, but routine, reliable de novo sequencing without database assistance is not standard (7–10).A primary means to facilitate de novo spectral interpretation is the simple annotation of m/z peaks in tandem mass spectra as either N- or C-terminal. We and others have investigated this seemingly simple first step. Real-world spectra, however, are complex. Difficulties often arise in determining the charge state of the fragment or in differentiating between fragment ions and peaks arising from neutral loss, internal fragmentation, or spectral noise, both electronic and chemical. Several strategies have focused on product ion annotation. These approaches have included manipulation of the N-terminus basicity combined with electron transfer dissociation (ETD)¹ (11–13). This approach can yield mostly N-terminal fragments for peptides having only two charges. However, it requires both ETD and the protease LysN. Other methods have used differential labeling of N- and C-terminal peptides to shift either one or the other product ion series, by either metabolic or chemical means (14–18). Metabolic incorporation of amino acids is an efficient method of introducing distinctive labels that eliminates in vitro labeling, but this method requires that the sample be amenable to cell culture (19, 20). Additionally, it may be difficult to achieve complete labeling in complex systems. Several other approaches used to introduce heavy isotopes onto one terminus have been investigated, including trypsin digestion in ¹⁸O water (21–23), differential isotopic esterification (24, 25), derivatization of the C-terminal carboxylate by p-bromophenethylamine (8, 26), N-terminal derivatization with sulfonic acid groups (27, 28), and formaldehyde labeling via reductive amination (29–31). These chemical modifications are introduced after cell lysis, often immediately prior to analysis. Although chemical labeling strategies can be used with a variety of samples, difficulties can arise from differences in labeling efficiency between samples, and often a clean-up step is required following labeling, which may lead to sample loss. No matter the labeling method, in this regime, the two precursors must be separately isolated, fragmented, and analyzed either together or separately. The recognition and selection of the broadly spaced doublet in real time also are necessary. These requirements have limited the utility of these approaches. Our own laboratory discovered that the c- and ^●z-type product ions generated from either electron capture dissociation or ETD have distinct chemical formulae and therefore can always be distinguished based on accurate mass alone (32). The problem with this approach is that extremely high mass accuracy (<500 ppb) is required in order to distinguish these product ion types above ∼600 Da in mass. Thus, the majority of the product ions within a spectrum cannot be readily mapped to either terminus with high confidence.Despite these difficulties, we assert that robust de novo sequencing methodology would benefit greatly from a simple method that could be used to distinguish N- and C-terminal product ions with high accuracy and precision. Ideally, the approach would work regardless of the choice of proteolytic enzyme or dissociation method. Recently, we described a new technology for protein quantification called neutron encoding (NeuCode) (33). NeuCode embeds millidalton mass differences into peptides and proteins by exploiting the mass defect induced by differences in the nuclear binding energies of the various stable isotopes of common elements such as C, N, H, and O. For example, consider the amino acid lysine, which has eight additional neutrons (+8 Da). One way to synthesize this amino acid is to add six ¹³C atoms and two ¹⁵N atoms (+8.0142 Da). Another isotopologue could be constructed by adding eight ²H atoms (+8.0502). These two isotopologues differ by only 36 mDa; peptide precursors containing both of these amino acids would appear as a single, unresolved precursor m/z peak at a mass resolving power of less than ∼100,000. However, under high resolving powers (i.e. greater than ∼100,000 at m/z 400), this doublet is resolved. We first developed this NeuCode concept in the context of metabolic labeling, akin to stable isotope labeling with amino acids in cell culture (SILAC), except that instead of the precursor partners being separated by 4 to 8 Da, they are separated by only 6 to 40 mDa. For quantitative purposes, NeuCode promises to deliver ultraplexed SILAC (>12) without increasing spectral complexity.We reasoned that the isotopologues of Lys that permit NeuCode SILAC would generate a distinct fingerprint on C-terminal product ions. Specifically, peptides that have been labeled with NeuCode SILAC and digested with LysC uniformly contain Lys at the C terminus. Upon MS/MS, all C-terminal product ions should present as doublets (with duplex NeuCode), whereas N-terminal products will be detected as a single m/z peak. The very close m/z spacing of the NeuCode SILAC partners will ensure that each partner is always co-isolated and that the signatures are visible only upon high-resolving-power mass analysis. Here we investigate the combination of NeuCode SILAC and high-resolving-power MS/MS analysis to allow the straightforward identification of C-terminal product ions.

Sample Preparation

Saccharomyces cerevisiae strain BY4741 Lys1Δ was grown in defined synthetic complete (SC, Sunrise Science, San Diego, CA) drop-out media with either heavy 6C¹³/2N¹⁵ lysine (+8.0142 Da, Cambridge Isotopes, Tewksbury, MA), or heavy 8D (+8.0502 Da, Cambridge Isotopes). Cells were propagated to a minimum of 10 doublings. At mid-log phase, cells were harvested via centrifugation at 3,000 × g for 3 min and then washed three times with chilled double distilled H₂O. Cell pellets were resuspended in 5 ml lysis buffer (50 mm Tris pH 8, 8 m urea, 75 mm sodium chloride, 100 mm sodium butyrate, 1 mm sodium orthovanadate, protease and phosphatase inhibitor tablet), and protein was extracted via glass bead milling (Retsch, Haan, Germany). Protein concentration was measured via BCA (Pierce). Cysteines in the yeast lysate were reduced with 5 m dithiothreitol at ambient temperature for 30 min, alkylated with 15 mm iodoacetamide in the dark at ambient temperature for 30 min, and then quenched with 5 mm dithiothreitol. 50 mm tris (pH 8.0) was used to dilute the urea concentration to 4 m. Proteins were digested with LysC (1:50 enzyme:protein ratio) at ambient temperature for 16 h. The digestion was quenched with TFA and desalted with a tC18 Sep-Pak (Waters, Etten-Leur, The Netherlands). Samples were prepared by mixing 6C¹³/2N¹⁵ (+8.0412 Da) and 8D (+8.0502 Da) labeled peptides in 1:1 ratios by mass. For strong cation exchange fractionation, peptides were dissolved in 400 μl of strong cation exchange buffer A (5 mm KH₂PO₄ and 30% acetonitrile; pH 2.65) and injected onto a polysulfoethylaspartamide column (9.4 mm × 200 mm; PolyLC) attached to a Surveyor LC quarternary pump (Thermo Electron, West Chester, PA) operating at 3 ml/min. Peptides were detected by photodiode array detector (Thermo Electron, West Chester, PA). Fractions were collected every 2 min starting at 10 min into the following gradient: 0–2 min at 100% buffer A, 2–5 min at 0%–15% buffer B (5 mm KH₂PO₄, 30% acetonitrile, and 350 mm KCl (pH 2.65)), and 5–35 min at 15%–100% buffer B. Buffer B was held at 100% for 10 min. Finally, the column was washed with buffer C (50 mm KH₂PO₄ and 500 mm KCl (pH 7.5)) and water before recalibration. Fractions were collected by hand every 2 to 3 min starting at 10 min into the gradient and were lyophilized and desalted with a tC18 Sep-Pak (Waters).

LC-MS/MS

Samples were loaded onto a 15-cm-long, 75-μm capillary column packed with 5 μm Magic C18 (Michrom, Auburn, CA) particles in mobile phase A (0.2% formic acid in water). Peptides were eluted with mobile phase B (0.2% formic acid in acetonitrile) over a 120-min gradient at a flow rate of 300 nl/min. Eluted peptides were analyzed by an Orbitrap Elite mass spectrometer. For the nonfractionated samples, mass spectrometer instrument methods comprised one MS¹ scan followed by data-dependent MS² scans of the five most intense precursors. A survey MS¹ scan was performed by the Orbitrap at 30,000 resolving power to identify precursors to sample for tandem mass spectrometry, and this was followed by an additional MS¹ scan at 480,000 resolving power (at m/z 400; actual mass resolving power of 470,700). Data-dependent tandem mass spectrometry was performed via beam-type collisional activated dissociation (HCD) in the Orbitrap at a resolving power of 15,000, 60,000, 120,000, or 240,000 and a collision energy of 30. Preview mode was enabled, and precursors of unknown charge or with a charge of +1 were excluded from MS² sampling. For experiments comparing the duty cycle and resolving power required in order to distinguish y-ion doublets, MS¹ and MS² target ion accumulation values were set to 5 × 10⁵ and 5 × 10⁴, respectively. For all other experiments, MS¹ target accumulation values were set to 1 × 10⁶ and MS² accumulation values were set to 4 × 10⁵. Dynamic exclusion was set to 30 s for −0.55 m/z and +2.55 m/z of selected precursors. For ETD analysis, data-dependent top-five mass spectrometry was performed at a resolving power of 240,000 (m/z 400; actual MS² mass resolving power of 271,000) (34). ETD accumulation values were set to 1 × 10⁶ for MS¹ target accumulation and 4 × 10⁵ for MS² target accumulation. The fluoranthene reaction time was set to 100 ms. For the high-pH strong cation exchange fractions, data-dependent tandem mass spectrometry was performed via HCD at a resolving power of either 60,000 or 120,000 and a collision energy of 30. Preview mode was enabled, and precursors of unknown charge or with a charge of +1 were excluded from MS² sampling. MS¹ targets were set to 1 × 10⁶, and MS² accumulation values were set to 4 × 10⁵. Dynamic exclusion was set to 45 s for −0.55 m/z and +2.55 m/z of selected precursors. Analysis by use of a wide isolation window was performed on an Orbitrap Fusion. MS¹ analysis was performed at 450,000 resolving power (m/z 200), and MS² analysis was performed at 120,000 resolving power (m/z 400). Data-dependent top-N mass spectrometry was performed, with precursors selected from sequential 25-Da windows. HCD was performed twice on the same precursor, first by use of a quadrupole isolation width of 0.7 m/z for peptide identification, and then using 25 m/z quadrupole isolation. Fragment ions were analyzed in the Orbitrap at a mass resolving power of 120,000 (m/z 400). MS¹ and MS² target accumulation values were set to 2 × 10⁵ and 5 × 10⁴, respectively.

Data Analysis

Thermo.raw files were converted to searchable DTA text files using the Coon OMSSA Proteomic Analysis Software Suite (COMPASS) (35). DTA files containing exclusively y-ions were generated using an in-house algorithm. DTA files were searched against the UniProt yeast database (version 132) with Lys-C specificity using the Open Mass Spectrometry Search Algorithm (OMSSA), version 2.1.9 (36). Methionine oxidation was searched as a variable modification. Cysteine carbamidomethylation and the mass shift imparted by the lysine isotopolgues were searched as fixed modifications. For MS² scans performed at a resolving power of 60,000, 120,000, or 240,000, a shift of +8.0142, representing the mass shift of the ¹³C₆¹⁵N₂ isotopologue, was searched. For MS² scans performed at 15,000 resolving power, the average shift of the ¹³C₆¹⁵N₂ and ⁸H₂ isotopologues (+8.0322) was searched. For all analyses, the precursor mass was obtained from the 480,000 MS scan. The precursor mass tolerance was defined as 50 ppm, and the fragment ion mass tolerance was set to 0.01 Da. A histogram of precursor mass error at different search tolerances is presented in supplemental Fig. S1. Using the COMPASS software suite, obtained search results were filtered to 1% FDR based on E-values. y-ion doublets were extracted from raw files using an in-house algorithm explained in the supplemental information. Briefly, an ensemble of three different machine learning models was used to score each MS/MS spectral peak for C-terminal product ion prediction. To train our ensemble learner to correctly distinguish C-terminal product ion peaks from N-terminal product ion peaks and noise peaks within our experimental MS/MS spectra, we generated a representative training set of spectral data. Instances used for training and test sets were peaks acquired only from MS/MS spectra associated with a peptide identification. Peaks with a signal-to-noise value of less than 5 were not used. Feature information for each training/testing instance was extracted from raw spectral data. Seven MS/MS spectral features were selected to generate training and test set data: (1) “has doublet” (evaluated as “true” only if a spectral peak could be found at the predicted m/z of the peak''s “heavy” partner), (2) “signal-to-noise” (discretized using a scale of 1–5 based on the peak''s signal-to-noise value), (3) “is isotope,” (4) “is neutral loss,” (5) “number of isotopes,” (6) “number of doublet isotopes,” and (7) “has neutral loss.”To evaluate NeuCode SILAC labeling for automated de novo sequencing, PepNovo⁺ (8) was benchmarked on y-ion predicted spectra. First, a set of identified spectra from 13,832 unique peptides (>7,400 per precursor charge 2–3) was produced to train PepNovo⁺ so it could learn features such as the relative peak height ranks of b/y-ions and the probability of noise at each mass interval. These training spectra were acquired under the 11 NeuCode yeast strong cation exchange fractions prepared as described above. Thermo raw files were converted into mzXML format using ProteoWizard v2.2.2828 (with peak-picking turned on) and identified by MS-GF⁺ v9358 (37) at a 1% spectrum-level FDR against the UniProt yeast database (plus isoforms), v20110729. A fixed modification of K⁺8.0142 was imposed along with variable modifications of oxidized Met and deamidated Asn/Gln. All MS/MS scans were searched with a 50-ppm precursor mass tolerance, the high-accuracy LTQ instrument setting, the HCD fragmentation setting, and one allowed missed Lys-C cleavage.Thermo.raw files were also converted into DTA spectra as before, except the in-house algorithm for selecting y-ion doublets was slightly altered to boost the peak height of predicted y-ions above that of other peaks (the cumulative peak height was equal to the sum of the monoisotopic doublet peaks, all isotopic doublet peaks, and two times the peak height of the base peak) and to convert their m/z to charge one. Remaining peaks not predicted to be y-ions were converted to charge one by a previously described MS/MS deconvolution tool (38). Deconvoluted DTA spectra that originated from identified MS/MS scans were then paired with the MSGF⁺ peptide IDs and passed to PepNovo⁺ for training. The resulting PepNovo⁺ scoring model lacked the rank-boosting component (39), which requires identified spectra from >100,000 unique peptides per precursor charge state and extensive modification of the PepNovo⁺ source code to train. Still, the model was sufficient to perform de novo peptide sequencing on the y-ion predicted spectra. PepNovo⁺ was also run on the raw MS/MS scans (mzXML spectra converted to MGF with all MS/MS peaks converted to charge one) by use of a previously trained HCD scoring model that also lacks the rank-boosting component (40). The following PepNovo⁺ parameters were set at all stages of training and benchmarking: fixed modification of K⁺8.0142; variable modifications of oxidized Met and deamidated Asn; 0.01-Da fragment mass tolerance; use of spectrum precursor charge; and use of spectrum precursor m/z.