Over the past decades, proteomics has become increasingly important and a heavily discussed topic. The identification of intact proteins remains a major focus in this field. While most intact proteins are analyzed using high-resolution mass spectrometry, identifying them through low-resolution mass spectrometry continues to pose challenges. In our study, we investigated the capability of identifying various intact proteins using collision-induced dissociation (CID) and electron transfer without dissociation (ETnoD). Using myoglobin as our test protein, stable product ions were generated with CID, and the identities of the product ions were identified with ETnoD. ETnoD uses a short activation time (AcT, 5 ms) to create sequential charge-reduced precursor ion (CRI). The charges of the fragments and their sequences were determined with corresponding CRI. The product ions can be selected for subsequent CID (termed CIDn) combined with ETnoD for further sequence identification and validation. We refer to this method as CIDn/ETnoD. The use of a multistage CID activation (CIDn) and ETnoD protocol has been applied to several intact proteins to obtain multiple sequence identifications.

Two Gram-stain-negative, rod-shaped, non-motile, strictly aerobic strains, forming yellow colonies and designated F6058T and S2608T, were isolated from marine sediment collected in Weihai, PR China. Both strains grow at 4-40 °C (optimum, 30-33 °C), pH 6.0-7.5 (optimum, pH 6.5) and in the presence of 0-7.0 % (w/v) NaCl. The optimum NaCl concentrations for strains F6058T and S2608T were 2.0 % and 2.5 %, respectively. Phylogenetic analysis of the 16S rRNA gene sequences indicated that strains F6058T and S2608T share an evolutionary lineage with members of the genus Aequorivita. The isolates exhibited a 16S rRNA gene sequence similarity of 96.7 % to each other. Strains F6058T exhibited the highest 16S rRNA gene sequence similarity to Aequorivita xiaoshiensis F64183T (98.8 %), and S2608T was most similar to Aequorivita capsosiphonis A71T (96.9 %). Iso-C15:0, anteiso-C15:0 and iso-C17:0 3-OH were the major fatty acids of strains F6058T and S2608T. The sole respiratory quinone of both isolates was menaquinone 6 (MK-6). The polar lipid profiles of the isolates both consisted of phosphatidylethanolamine and phosphoglycolipids; however, strain F6058T exhibited one glycolipid, one aminolipid and two unidentified polar lipids, and strain S2608T also had two glycolipids and one unidentified polar lipid. The DNA G+C contents of strains F6058T and S2608T were 34.6 % and 37.7 mol%, respectively. Based on their phenotypic, chemotaxonomic and genomic characteristics, strains F6058T and S2608T were considered to represent novel species of the genus Aequorivita, for which the names Aequorivita sediminis sp. nov. and Aequorivita marina sp. nov. were proposed. The type strains are F6058T (=KCTC 92653T=MCCC 1H01358T) and S2608T (KCTC 92652T=MCCC 1H01361T).

The analysis of the structures of glycans present on glycoproteins is an essential component for determining glycoprotein function; however, detailed glycan structural assignment on glycopeptides from proteomics mass spectrometric data remains challenging. Glycoproteomic analysis by mass spectrometry currently can provide significant, yet incomplete, information about the glycans present, including the glycan monosaccharide composition and in some circumstances the site(s) of glycosylation. Advancements in mass spectrometric resolution, using high-mass accuracy instrumentation and tailored MS/MS fragmentation parameters, coupled with a dedicated definition of diagnostic fragmentation ions have enabled the determination of some glycan structural features, or glycotopes, expressed on glycopeptides. Here we present a collation of diagnostic glycan fragments produced by traditional positive-ion-mode reversed-phase LC-ESI MS/MS proteomic workflows and describe the specific fragmentation energy settings required to identify specific glycotopes presented on N- or O-linked glycopeptides in a typical proteomics MS/MS experiment.

A Gram-stain-negative, orange-yellow, rod-shaped bacterium, designated strain SCSIO 19198T, was isolated from sediment of the Haima cold seep in the South China Sea, PR China. The strain was aerobic and non-motile. Growth of strain SCSIO 19198T occurred at pH 7-9 (optimum, pH 7), 15-37 °C (optimum, 25-32 °C) and with 3-8 % (w/v) NaCl (optimum, 3-6 % NaCl). Phylogenetic analyses based on 16S rRNA sequences revealed that strain SCSIO 19198T belonged to the genus Hwangdonia, having the highest similarity to Hwangdonia seohaensis HD-3T (98.35 %), followed by Algibacter aquimarinus KYW589T (95.17 %) and Gelatiniphilus marinus GYP-24T (94.89 %). The DNA G+C content was 35.92 mol%. The average nucleotide identity value between the genome of strain SCSIO 19198T and that of H. seohaensis HD-3T was 88.49 %. The digital DNA-DNA hybridization value between strain SCSIO 19198T and H. seohaensis HD-3T was 36 %. The major fatty acids (>10 %) of strain SCSIO 19198T were iso-C15 : 0, iso-C15 : 1 G, summed feature 3 (C16 : 1  ω6c/C16 : 1 ω7c) and anteiso-C15 : 0. MK-6 was the only detected respiratory quinone. The polar lipids of strain SCSIO 19198T included phosphatidylethanolamine, two aminolipids, glycolipid and two unidentified lipids. The phenotypic, phylogenetic, chemotaxonomic and genomic data clearly suggest that strain SCSIO 19198T represents a novel species of the genus Hwangdonia, for which the name Hwangdonia lutea sp. nov. is proposed. The type strain is SCSIO 19198T (=MCCC 1K08674T=KCTC 102078T).

Mastophorus muris (Gmelin, 1790) is a globally distributed parasitic nematode of broad range mammals. The taxonomy within the genus Mastophorus and the cryptic diversity among the genus are controversial among taxonomists. This study provides a detailed morphological description of M. muris from Mus musculus combined with a molecular phylogenetic approach. Moreover, descriptions and molecular data of M. muris from non-Mus rodents and wildcats complement our findings and together provide new insights into their taxonomy. The analysis of M. muris was based on light microscopy and scanning electron microscopy. The morphological description focused on the dentition pattern of the two trilobed pseudolabia. Additionally, we described the position of the vulva, arrangement of caudal pairs of papillae, spicules and measured specimens from both sexes and the eggs. For the molecular phylogenetic approach, we amplified the small subunit ribosomal RNA gene and the internal transcribed spacer, and the cytochrome c oxidase subunit 1. Mastophorus morphotypes based on dentition patterns and phylogenetic clustering indicate a subdivision of the genus in agreement with their host. We recognize two groups without a change to formal taxonomy: One group including those specimens infecting Mus musculus, and the second group including organisms infecting non-Mus rodents. Our genetic and morphological data shed light into the cryptic diversity within the genus Mastopohorus. We identified two host-associated groups of M. muris. The described morphotypes and genotypes of M. muris allow a consistent distinction between host-associated parasites.

Ticks, blood-sucking ectoparasites, spread diseases to humans and animals. Haemaphysalis longicornis is a significant vector for tick-borne diseases in medical and veterinary contexts. Identifying protective antigens in H. longicornis for an anti-tick vaccine is a key tick control strategy. Enolase, a multifunctional protein, significantly converts D-2-phosphoglycerate and phosphoenolpyruvate in glycolysis and gluconeogenesis in cell cytoplasm. This study cloned a complete open reading frame (ORF) of enolase from the H. longicornis tick and characterized its transcriptional and silencing effect. We amplified the full-length cDNA of the enolase gene using rapid amplification of cDNA ends. The complete cDNA, with an ORF of 1,297 nucleotides, encoded a 432-amino acid polypeptide. Enolase of the Jeju strain H. longicornis exhibited the highest sequence similarity with H. flava (98%), followed by Dermacentor silvarum (82%). The enolase motifs identified included N-terminal and C-terminal regions, magnesium binding sites, and several phosphorylation sites. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that enolase mRNA transcripts were expressed across all developmental stages of ticks and organs such as salivary gland and midgut. RT-PCR showed higher transcript levels in syn-ganglia, suggesting that synganglion nerves influence enolase,s role in tick salivary glands. We injected enolase double-stranded RNA into adult unfed female ticks, after which they were subsequently fed with normal unfed males until they spontaneously dropped off. RNA interference significantly (P<0.05) reduced feeding and reproduction, along with abnormalities in eggs (no embryos) and hatching. These findings suggest enolase is a promising target for future tick control strategies.

Voltage-Dependent Anion Channel isoforms (VDAC1, VDAC2, and VDAC3) are relevant components of the outer mitochondrial membrane (OMM) and play a crucial role in regulation of metabolism and in survival pathways. As major players in the regulation of cellular metabolism and apoptosis, VDACs can be considered at the crossroads between two broad families of pathologies, namely, cancer and neurodegeneration, the former being associated with elevated glycolytic rate and suppression of apoptosis in cancer cells, the latter characterized by mitochondrial dysfunction and increased cell death. Recently, we reported the characterization of the oxidation pattern of methionine and cysteines in rat and human VDACs showing that each cysteine in these proteins is present with a preferred oxidation state, ranging from the reduced to the trioxidized form, and such an oxidation state is remarkably conserved between rat and human VDACs. However, the presence and localization of disulfide bonds in VDACs, a key point for their structural characterization, have so far remained undetermined. Herein we have investigated by nanoUHPLC/High-Resolution nanoESI-MS/MS the position of intramolecular disulfide bonds in rat VDAC2 (rVDAC2), a protein that contains 11 cysteines. To this purpose, extraction, purification, and enzymatic digestions were carried out at slightly acidic or neutral pH in order to minimize disulfide bond interchange. The presence of six disulfide bridges was unequivocally determined, including a disulfide bridge linking the two adjacent cysteines 4 and 5, a disulfide bridge linking cysteines 9 and 14, and the alternative disulfide bridges between cysteines 48, 77, and 104. A disulfide bond, which is very resistant to reduction, between cysteines 134 and 139 was also detected. In addition to the previous findings, these results significantly extend the characterization of the oxidation state of cysteines in rVDAC2 and show that it is highly complex and presents unusual features. Data are available via ProteomeXchange with the identifier PXD044041.

Two Gram-negative bacteria, designated as strains LF1T and HM2-2T, were isolated from an artificial pond in a honey farm at Hoengseong-gun, Gangwon-do, Republic of Korea. The 16S rRNA sequence analysis results revealed that strain LF1T belonged to the genus Lysobacter and had the highest sequence similarity to Lysobacter niastensis GH41-7T (99.0 %), Lysobacter panacisoli CJ29T (98.9 %), and Lysobacter prati SYSU H10001T (98.2 %). Its growth occurred at 20-37 °C, at pH 5.0-12.0, and in the presence of 0-2% NaCl. The major fatty acids were iso-C15 : 0, iso-C16 : 0, and summed feature 9 (iso-C17 : 1  ω9c and/or C16 : 0 10-methyl). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The DNA G+C content was 67.5 mol%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain LF1T and species of the genus Lysobacter were 79.1-84.4% and 22.0-27.5 %, respectively. The 16S rRNA sequence analysis results revealed that strain HM2-2T belonged to the genus Limnohabitans and was most closely related to Limnohabitans planktonicus II-D5T (98.9 %), Limnohabitans radicicola JUR4T (98.4%), and Limnohabitans parvus II-B4T (98.4 %). Its growth occurred at 10-35 °C, at pH 5.0-11.0, and in the presence of 0-2% NaCl. The major fatty acids were C16 : 0 and summed feature 3 (C16 : 1  ω7c/C16 : 1  ω6c). The major polar lipid was phosphatidylethanolamine. The DNA G+C content was 59.9 mol%. The ANI and dDDH values between strain HM2-2T and its closely related strains were 75.1-83.0% and 20.4-26.4 %, respectively. Phenotypic, genomic, and phylogenetic data revealed that strains LF1T and HM2-2T represent novel species in the genera Lysobacter and Limnohabitans, for which the names Lysobacter stagni sp. nov. and Limnohabitans lacus sp. nov. are proposed, respectively. The type strain of Lys. stagni is LF1T (=KACC 23251T=TBRC 17648T), and that of Lim. lacus is HM2-2T (=KACC 23250T=TBRC 17649T).

It has been the aim of this study to molecular-taxonomically identify 15 Beauveria isolates collected from different geographical regions and insect hosts in Argentina and to investigate the levels of inter- and intra-specific diversity across this set of isolates. Based on phylogenetic analyses of EF1A-RPB1-RPB2 concatenated genes and BLOC markers, all Beauveria strains were identify as Beauveria bassiana. Within the B. bassiana clades of both phylogenies, isolates from Argentina were not clustered according to geographic origin or host. The 15 fungal isolates were further analyzed by PCR amplification of the intron insertion hot spot region of the nuclear 28S rRNA encoding sequence. By intron sequence and position, seven different group-I intron combinations termed variants A, B1, B2, C, D, E and F were found in the 15 isolates under study. Variants B1/B2 consisting of a single 28Si2 intron were found in ten isolates, whereas variant A occurred twice and variants C through F were unique across the set of isolates under study. The determination of the different introns and intron combinations in the 28S rRNA gene is a powerful tool for achieving infraspecific differentiation of B. bassiana isolates from Argentina.

A Gram-negative, rod-shaped, non-motile and strictly aerobic strain, designated NBU2979T, was isolated from a coastal mudflat located on Meishan Island in the East China Sea. Strain NBU2979T grew optimally at 32 °C, with 2.0 % NaCl (w/v) and at pH 7.0-7.5. The predominant fatty acid (>10 %) was iso-C15 : 0. The major polar lipids included phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, phosphatidyldimethylethanolamine, phosphatidylcholine, an unidentified glycolipid, two unidentified aminophospholipids, an unidentified phospholipid and an unidentified lipid. The only respiratory quinone was ubiquinone-8. Comparative analysis of 16S rRNA gene sequences showed that strain NBU2979T exhibited highest similarity to Marinicella sediminis F2T (98.0 %), Marinicella marina S1101T (97.5 %), Marinicella litoralis KMM 3900T (96.6 %), Marinicella rhabdoformis 3539T (95.5 %), Marinicella pacifica sw153T (95.2 %) and Marinicella gelatinilytica S6413T (94.9 %). Phylogenetic analyses indicated that strain NBU2979T clustered with the genus Marinicella and was closely related to strain M. sediminis F2T. The average nucleotide identity and digital DNA-DNA hybridization values between strain NBU2979T and related species of genus Marinicella were well below the threshold limit for prokaryotic species delineation. The DNA G+C content of strain NBU2979T was 51.6 mol%. Based on its phenotypic, chemotaxonomic and genotypic data, strain NBU2979T (=KCTC 82911T=MCCC 1K06402T) is considered to be a representative of a novel species in the genus Marinicella, for which the name Marinicella meishanensis sp. nov. is proposed.