Middle-down hybrid chromatography/tandem mass spectrometry workflow for characterization of combinatorial post-translational modifications in histones.

We present an integrated middle-down proteomics platform for sensitive mapping and quantification of coexisting PTMs in large polypeptides (5-7 kDa). We combined an RP trap column with subsequent weak cation exchange-hydrophilic interaction LC interfaced directly to high mass accuracy ESI MS/MS using electron transfer dissociation. This enabled automated and efficient separation and sequencing of hypermodified histone N-terminal tails for unambiguous localization of combinatorial PTMs. We present Histone Coder and IsoScale software to extract, filter, and analyze MS/MS data, including quantification of cofragmenting isobaric polypeptide species. We characterized histone tails derived from murine embryonic stem cells knockout in suppressor of zeste12 (Suz12(-/-) ) and quantified 256 combinatorial histone marks in histones H3, H4, and H2A. Furthermore, a total of 713 different combinatorial histone marks were identified in purified histone H3. We measured a seven-fold reduction of H3K27me2/me3 (where me2 and me3 are dimethylation and trimethylation, respectively) in Suz12(-) (/) (-) cells and detected significant changes of the relative abundance of 16 other single PTMs of histone H3 and other combinatorial marks. We conclude that the inactivation of Suz12 is associated with changes in the abundance of not only H3K27 methylation but also multiple other PTMs in histone H3 tails.

Deconvolution of mixture spectra and increased throughput of peptide identification by utilization of intensified complementary ions formed in tandem mass spectrometry.

A cornerstone of mass spectrometry based proteomics is to relate with high statistical significance experimentally obtained tandem mass spectrometry (MS/MS) data to peptide sequences from a protein database. Most sequence specific fragment ions in MS/MS spectra are represented by a subset of complementary ion pairs. Here, we investigated the reliabilities of complementary ion pairs formed in CAD and CAD/ETD MS/MS and developed a reliability-based approach of intensification of ion signals of complementary pairs prior to database searching. In a large-scale proteomics experiment using high-resolution orbitrap mass spectrometry, an increase in the number of peptide identifications was obtained relative to the original CAD MS/MS spectra when intensified golden complementary (+18.6%) and CAD complementary pairs (+17.2%) were submitted to the Mascot search engine. This also exceeded the results obtained by deisotoping/deconvolution of CAD MS/MS spectra. A novel approach for extracting sequence-specific fragment ions of co-isolated peptides was developed based on the complementarity rules. This technique demonstrated an impressive gain of 42.4% more peptide identifications as compared with the use of the initial data set.

Self-pacing ameliorates recall deficit when listening to vocoded discourse: a cochlear implant simulation.

Introduction: In spite of its apparent ease, comprehension of spoken discourse represents a complex linguistic and cognitive operation. The difficulty of such an operation can increase when the speech is degraded, as is the case with cochlear implant users. However, the additional challenges imposed by degraded speech may be mitigated to some extent by the linguistic context and pace of presentation. Methods: An experiment is reported in which young adults with age-normal hearing recalled discourse passages heard with clear speech or with noise-band vocoding used to simulate the sound of speech produced by a cochlear implant. Passages were varied in inter-word predictability and presented either without interruption or in a self-pacing format that allowed the listener to control the rate at which the information was delivered. Results: Results showed that discourse heard with clear speech was better recalled than discourse heard with vocoded speech, discourse with a higher average inter-word predictability was better recalled than discourse with a lower average inter-word predictability, and self-paced passages were recalled better than those heard without interruption. Of special interest was the semantic hierarchy effect: the tendency for listeners to show better recall for main ideas than mid-level information or detail from a passage as an index of listeners' ability to understand the meaning of a passage. The data revealed a significant effect of inter-word predictability, in that passages with lower predictability had an attenuated semantic hierarchy effect relative to higher-predictability passages. Discussion: Results are discussed in terms of broadening cochlear implant outcome measures beyond current clinical measures that focus on single-word and sentence repetition.

Strategic Pauses Relieve Listeners from the Effort of Listening to Fast Speech: Data Limited and Resource Limited Processes in Narrative Recall by Adult Users of Cochlear Implants.

Speech that has been artificially accelerated through time compression produces a notable deficit in recall of the speech content. This is especially so for adults with cochlear implants (CI). At the perceptual level, this deficit may be due to the sharply degraded CI signal, combined with the reduced richness of compressed speech. At the cognitive level, the rapidity of time-compressed speech can deprive the listener of the ordinarily available processing time present when speech is delivered at a normal speech rate. Two experiments are reported. Experiment 1 was conducted with 27 normal-hearing young adults as a proof-of-concept demonstration that restoring lost processing time by inserting silent pauses at linguistically salient points within a time-compressed narrative ("time-restoration") returns recall accuracy to a level approximating that for a normal speech rate. Noise vocoder conditions with 10 and 6 channels reduced the effectiveness of time-restoration. Pupil dilation indicated that additional effort was expended by participants while attempting to process the time-compressed narratives, with the effortful demand on resources reduced with time restoration. In Experiment 2, 15 adult CI users tested with the same (unvocoded) materials showed a similar pattern of behavioral and pupillary responses, but with the notable exception that meaningful recovery of recall accuracy with time-restoration was limited to a subgroup of CI users identified by better working memory spans, and better word and sentence recognition scores. Results are discussed in terms of sensory-cognitive interactions in data-limited and resource-limited processes among adult users of cochlear implants.

Reduction in database search space by utilization of amino acid composition information from electron transfer dissociation and higher-energy collisional dissociation mass spectra.

With high-mass accuracy and consecutively obtained electron transfer dissociation (ETD) and higher-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS), reliable (≥97%) and sensitive fragment ions have been extracted for identification of specific amino acid residues in peptide sequences. The analytical benefit of these specific amino acid composition (AAC) ions is to restrict the database search space and provide identification of peptides with higher confidence and reduced false negative rates. The 6706 uniquely identified peptide sequences determined with a conservative Mascot score of >30 were used to characterize the AAC ions. The loss of amino acid side chains (small neutral losses, SNLs) from the charge reduced peptide radical cations was studied using ETD. Complementary AAC information from HCD spectra was provided by immonium ions. From the ETD/HCD mass spectra, 5162 and 6720 reliable SNLs and immonium ions were successfully extracted, respectively. Automated application of the AAC information during database searching resulted in an average 3.5-fold higher confidence level of peptide identification. In addition, 4% and 28% more peptides were identified above the significance level in a standard and extended search space, respectively.

Automated and high confidence protein phosphorylation site localization using complementary collision-activated dissociation and electron transfer dissociation tandem mass spectrometry.

Reversible protein phosphorylation plays a critical role in cell signaling and is responsible for the regulation of many biological processes in most living organisms. The low stoichiometry of protein phosphorylation requires sensitive analysis by tandem mass spectrometry. However, incomplete peptide fragmentation and the loss of labile phosphate groups complicate identification of the site of the phosphate motif. Here, we have implemented and evaluated a novel approach for phospho-site localization by the combined use of peptide tandem mass spectrometry data obtained using both collision-activated dissociation and electron transfer dissociation, an approach termed the Cscore. The scoring algorithm used in the Cscore was adapted from the widely used Ascore method. The analytical benefit of integrating the product ion information of both ETD and CAD data are evident by increased confidence in phospho-site localization and the number of assigned phospho-sites at a fixed false-localization rate. The average calculated Cscore from a large data set (>7000 phosphopeptide MS/MS spectra) was ∼32 compared to ∼23 and ∼17 for the Ascore using collision-activated dissociation (CAD) or electron transfer dissociation (ETD), respectively. Compared with the Ascore using either CAD or ETD, the Cscore identified up to 88% more phosphorylation sites. Using a phosphopeptide library revealed that the score threshold for obtaining a false-localization rate of 0.5% was lower for the Cscore than either the Ascore (CAD) or the Ascore (ETD).

A Sporadic Four-Year Hospital Outbreak of a ST97-IVa MRSA With Half of the Patients First Identified in the Community.

This study describes a sporadically occurring 4-year outbreak of methicillin-resistant Staphylococcus aureus (MRSA) originating from a surgical ward. Whole-genome sequencing (WGS) identified the outbreak clone as spa type t267, sequence type ST97, and SCCmec IVa. Prompted by the finding of four patients within 6 months in the same ward with this unusual MRSA type, an outbreak was suspected. Subsequent MRSA screening in the ward in February 2017 identified three-additional patients and two health care workers (HCWs) with t267/ST97-IVa. WGS linked these 9 isolates to 16 previous isolates in our WGS database and the outbreak thus included 23 patients and two HCWs. Twenty-one patients had a connection to the surgery ward during the period 2013-2017, but half of them had MRSA diagnosed in the community long after discharge. The community debut of several patients MRSA infections weeks to months after hospital discharge made the identification of a hospital source difficult and it was the SNP relatedness of the isolates that led us to identify the common denominator of hospitalization. An index patient was not identified, but our hypothesis is that HCWs with unrecognized long-term MRSA colonization could have caused sporadic nosocomial transmission due to intermittent breaches in infection prevention and control practice.

Draft genome sequence of a Kluyvera intermedia isolate from a patient with a pancreatic abscess.

The genus Kluyvera comprises potential pathogens that can cause many infections. This study reports a Kluyvera intermedia strain (FOSA7093) from a pancreatic cyst specimen from a long-term hospitalised patient. Whole-genome sequencing (WGS) of the K. intermedia isolate was performed and the strain was reported as sensitive to Danish-registered antibiotics although it had a fosA-like gene in the genome. There were nine contigs that aligned to a plasmid, and these contigs contained several heavy metal resistance gene homologues. Furthermore, a prophage was discovered in the genome. WGS represents an efficient tool for monitoring Kluyvera spp. and its role as a reservoir of multidrug resistance. Therefore, this susceptible K. intermedia genome has many characteristics that allow comparison of resistant K. intermedia that might be discovered in the future.

Whole Genome Sequencing of Danish Staphylococcus argenteus Reveals a Genetically Diverse Collection with Clear Separation from Staphylococcus aureus.

Staphylococcus argenteus (S. argenteus) is a newly identified Staphylococcus species that has been misidentified as Staphylococcus aureus (S. aureus) and is clinically relevant. We identified 25 S. argenteus genomes in our collection of whole genome sequenced S. aureus. These genomes were compared to publicly available genomes and a phylogeny revealed seven clusters corresponding to seven clonal complexes. The genome of S. argenteus was found to be different from the genome of S. aureus and a core genome analysis showed that ~33% of the total gene pool was shared between the two species, at 90% homology level. An assessment of mobile elements shows flow of SCCmec cassettes, plasmids, phages, and pathogenicity islands, between S. argenteus and S. aureus. This dataset emphasizes that S. argenteus and S. aureus are two separate species that share genetic material.

Electron transfer dissociation reveals changes in the cleavage frequencies of backbone bonds distant to amide-to-ester substitutions in polypeptides.

Interrogation of electron transfer dissociation (ETD) mass spectra of peptide amide-to-ester backbone bond substituted analogues (depsipeptides) reveals substantial differences in the entire backbone cleavage frequencies. It is suggested that the point permutation of backbone bonds leads to changes in the predominant ion structures by removal/weakening of specific hydrogen bonding. ETD responds to these changes by redistributing the cleavage frequencies of the peptide backbone bonds. In comparison, no distinction between depsi-/peptide was observed using collision-activated dissociation, which is consistent with a general unfolding and elimination of structural information of these ions. These results should encourage further exploration of depsipeptides for gas-phase structural characterization.