The ybiN gene of Escherichia coli encodes adenine-N6 methyltransferase specific for modification of A1618 of 23 S ribosomal RNA, a methylated residue located close to the ribosomal exit tunnel

N⁶-Methyladenosine 1618 of Escherichia coli 23 S rRNA is located in a cluster of modified nucleotides 12 Å away from the nascent peptide tunnel of the ribosome. Here, we describe the identification of gene ybiN encoding an enzyme responsible for methylation of A1618. Knockout of the ybiN gene leads to loss of modification at A1618. The modification is restored if ybiN knock-out strain has been co-transformed with a plasmid expressing the ybiN gene. On the basis of these results we suggest that ybiN gene should be renamed to rlmF in accordance with the accepted nomenclature for rRNA methyltransferases. Recombinant YbiN protein is able to methylate partially deproteinized 50 S ribosomal subunit, so-called 3.5 M LiCl core particle in vitro, but neither the completely assembled 50 S subunits nor completely deproteinized 23 S rRNA. Both lack of the ybiN gene and it's over-expression leads to growth retardation and loss of cell fitness comparative to the parental strain. It might be suggested that A1618 modification could be necessary for the exit tunnel interaction with some unknown regulatory peptides.     

Domain swapping in p13suc1 results in formation of native-like, cytotoxic aggregates

The field of protein aggregation has been occupied mainly with the study of beta-strand self-association that occurs as a result of misfolding and leads to the formation of toxic protein aggregates and amyloid fibers. However, some of these aggregates retain native-like structural and enzymatic properties suggesting mechanisms other than beta-strand assembly. p13suc1 is a small protein that can exist as a monomer or a domain-swapped dimer. Here, we show that, under native conditions, p13suc1 forms three-dimensional domain-swapped aggregates, and that these aggregates are cytotoxic. Thus, toxicity of protein aggregates is not only associated with beta-rich assemblies and amyloid fibers, involving non-native interactions, but it can be induced by oligomeric misassembly that maintains predominantly native-like interactions.     

Global changes in phospholipids identified by MALDI MS in rats with focal cerebral ischemia

Neuronal membrane phospholipids are highly affected by oxidative stress caused by ischemic injury. Thus, it is necessary to identify key lipid components that show changes during ischemia to develop an effective approach to prevent brain damage from ischemic injury. The recent development of MALDI imaging MS (MALDI IMS) makes it possible to identify phospholipids that change between damaged and normal regions directly from tissues. In this study, we conducted IMS on rat brains damaged by ischemic injury and detected various phospholipids that showed unique distributions between normal and damaged areas of the brain. Among them, we confirmed changes in phospholipids such as lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin by MALDI IMS followed by MS/MS analysis. These lipids were present in high concentrations in the brain and are important for maintenance of cellular structure as well as production of second messengers for cellular signal transduction. Our results emphasize the identification of phospholipid markers for ischemic injury and successfully identified several distinctly located phospholipids in ischemic brain tissue.     

Inhibition of hybrid- and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway

A mammalian N-acetylglucosamine (GlcNAc) transferase I (GnT I)-independent fucosylation pathway is revealed by the use of matrix-assisted laser desorption/ionization (MALDI) and negative-ion nano-electrospray ionization (ESI) mass spectrometry of N-linked glycans from natively folded recombinant glycoproteins, expressed in both human embryonic kidney (HEK) 293S and Chinese hamster ovary (CHO) Lec3.2.8.1 cells deficient in GnT I activity. The biosynthesis of core fucosylated Man5GlcNAc2 glycans was enhanced in CHO Lec3.2.8.1 cells by the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), leading to the increase in core fucosylated Man5GlcNAc2 glycans and the biosynthesis of a novel core fucosylated monoglucosylated oligomannose glycan, Glc1Man7GlcNAc2Fuc. Furthermore, no fucosylated Man9GlcNAc2 glycans were detected following inhibition of alpha-mannosidase I with kifunensine. Thus, core fucosylation is prevented by the presence of terminal alpha1-2 mannoses on the 6-antennae but not the 3-antennae of the trimannosyl core. Fucosylated Man5GlcNAc2 glycans were also detected on recombinant glycoprotein from HEK 293T cells following inhibition of Golgi alpha-mannosidase II with swainsonine. The paucity of fucosylated oligomannose glycans in wild-type mammalian cells is suggested to be due to kinetic properties of the pathway rather than the absence of the appropriate catalytic activity. The presence of the GnT I-independent fucosylation pathway is an important consideration when engineering mammalian glycosylation.     

Mass spectrometry images acylcarnitines,phosphatidylcholines, and sphingomyelin in MDA-MB-231 breast tumor models

The lipid compositions of different breast tumor microenvironments are largely unknown due to limitations in lipid imaging techniques. Imaging lipid distributions would enhance our understanding of processes occurring inside growing tumors, such as cancer cell proliferation, invasion, and metastasis. Recent developments in MALDI mass spectrometry imaging (MSI) enable rapid and specific detection of lipids directly from thin tissue sections. In this study, we performed multimodal imaging of acylcarnitines, phosphatidylcholines (PC), a lysophosphatidylcholine (LPC), and a sphingomyelin (SM) from different microenvironments of breast tumor xenograft models, which carried tdTomato red fluorescent protein as a hypoxia-response element-driven reporter gene. The MSI molecular lipid images revealed spatially heterogeneous lipid distributions within tumor tissue. Four of the most-abundant lipid species, namely PC(16:0/16:0), PC(16:0/18:1), PC(18:1/18:1), and PC(18:0/18:1), were localized in viable tumor regions, whereas LPC(16:0/0:0) was detected in necrotic tumor regions. We identified a heterogeneous distribution of palmitoylcarnitine, stearoylcarnitine, PC(16:0/22:1), and SM(d18:1/16:0) sodium adduct, which colocalized primarily with hypoxic tumor regions. For the first time, we have applied a multimodal imaging approach that has combined optical imaging and MALDI-MSI with ion mobility separation to spatially localize and structurally identify acylcarnitines and a variety of lipid species present in breast tumor xenograft models.     

基质辅助激光解析电离飞行时间质谱在医学真菌领域的应用进展

基质辅助激光解析电离飞行时间质谱（matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF MS）是近年来新兴的微生物检测技术,通过核糖体蛋白分析实现对真菌快速、准确鉴定。本文针对MALDI-TOF MS用于致病真菌鉴定、分类、体外抗真菌药物敏感性检测以及临床微生物样本直接检测等方面作一综述。     

一种SDS-PAGE与MALDI-TOF质谱联用的方法

以尿激酶的为材料,探索一种从ＳＤＳ－ＰＡＧＥ胶上回收蛋白 做ＭＡＬＤＩ－ＴＯＦ质谱的方法,所用的回收方法包括电洗脱、脱盐、除ＳＤＳ等过程,电洗脱用的是高盐阻断法,脱盐用的超滤技术,去除ＳＤＳ用的是冷丙沉淀法,结果证明,此方法至少对一些蛋白质是可行的。     

一种SDS-PAGE与MALDI-TOF质谱联用的方法

以尿激酶原为材料,探索一种从SDS-PAGE胶上回收蛋白质做MALDI-TOF质谱的方法.所用的回收方法包括电洗脱、脱盐、除SDS等过程.电洗脱用的是高盐阻断法,脱盐用的是超滤技术,去除SDS用的是冷丙酮沉淀法.结果证明,此方法至少对一些蛋白质（如尿激酶原和牛血清白蛋白）是可行的.     

Combination of Micropreparative Capillary Electrophoresis and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Peptide Analysis

Signal suppression is a problem in matrix-assisted laser desorption/ionization mass spectrometry of peptides prepared by capillary electrophoresis. Many common electrolytes that are efficient for separation, such as sodium phosphate, also are strongly suppressive during laser desorption/ionization. We have tested individual electrolytes for highest performance in each step of separation and collection, respectively. Suppression is not observed if citrate, trifluoroacetic acid, or hydrochloric acid is used for collection, while phosphate still can be employed in the capillary providing excellent resolution. Low concentrations of hydrochloric acid added to the sample/matrix mixture generate mass spectra with better ion intensities than if trifluoroacetic acid or citrate is used.     

Differential analysis of site-specific glycans on plasma and cellular fibronectins: application of a hydrophilic affinity method for glycopeptide enrichment

Isolation of glycopeptides utilizing hydrogen bonding between glycopeptide glycans and a carbohydrate-gel matrix in the organic phase is useful for site-specific characterization of oligosaccharides of glycoproteins, when combined with mass spectrometry. In this study, recovery of glycopeptides was improved by including divalent cations or increasing the organic solvent in the binding solution, without losing specificity, whereas it was still less effective for those with a long peptide backbone exceeding 50 amino acid residues. The method was then applied to the analysis of glycan heterogeneities at seven N-glycosylation sites in each of the plasma and cellular fibronectins (FNs). There was a remarkable site-specific difference in fucosylation between these isoforms; Asn1244 selectively escaped the global fucosylation of cellular FN, whereas only Asn1007 and Asn2108 of the plasma isoform underwent modification. In addition, a new O-glycosylation site was identified at Thr279 in the connecting segment between the fibrin- and heparin-binding domain and the collagen-binding domain, and the glycopeptide was reactive to a peanut agglutinin lectin. Considering that another mucin-type O-glycosylation site lies within a different connecting segment, the O-glycosylation of FN was suggested to play a significant role in segregating the neighboring domains and thus maintaining the topology of FN and the domain functions. In addition, the method was applied to apolipoprotein B-100 (apoB100) whose N-glycan structures at 17 of 19 potential sites have been reported, and characterized the remaining sites. The results also demonstrated that the enriched glycopeptide provides resources for site-specific analysis of oligosaccharides in glycoproteomics.     

Variation between strains of the cyanobacterium Microcystis aeruginosa isolated from a Portuguese river

AIMS: The aim of this study was to investigate toxicological differences between strains of the cyanobacterium Microcystis aeruginosa isolated from a potable water supply in the north of Portugal over a 2-month period. METHODS AND RESULTS: Twenty-six strains of M. aeruginosa were isolated, grown in pure culture, and tested using a range of techniques including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), ELISA and a PCR procedure targeting the genes implicated in the production of toxic microcystins. There was considerable variation with respect to the amounts of microcystin produced by each of the strains as measured by ELISA, with values ranging from 0.02 to 0.53% dry weight. The results of the MALDI-TOF MS analysis demonstrated the presence of several chemically distinct forms of microcystin as well as aeruginosins, anabaenopeptins and several other unidentified peptide-like compounds. CONCLUSIONS: The growth of individual strains that comprise bloom populations, with unique 'chemotypes' can potentially be an important factor affecting the toxicity of bloom populations. Molecular probes, targeting the genes responsible for microcystin production were shown to be useful for distinguishing between toxic and nontoxic strains and showed good agreement with the results obtained from the other analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study show that the analysis of cyanobacterial bloom populations at the subspecies (strain) level can potentially provide important information regarding the toxin-producing potential of a cyanobacterial bloom and could be used as an 'early warning' for toxic bloom development.     

ITS序列分析与MALDI-TOF MS质谱技术在丝状真菌鉴定中的应用

丝状真菌常用的鉴定方法为形态方法和基因鉴定方法,前者限于检验人员的知识和技能,后者操作繁琐,费用略昂贵,不适合常规开展。因此,寻找丝状真菌快速鉴定方法势在必行。本文采用VITEK MALDI-TOF MS（基质辅助激光解析电离时间飞行质谱）IVD数据库（3.0版本）对临床分离的254株丝状真菌进行鉴定,并以ITS（internal transcribed spacer 内转录间隔区）序列分析为标准,验证MALDI-TOF MS质谱技术鉴定丝状真菌的准确性。结果表明MALDI-TOF MS质谱技术可以对大部分丝状真菌实现快速、准确的鉴定,其中对毛癣菌属（100%）、毛孢子菌属（100%）、毛霉菌属（100%）、曲霉菌属（96.5%）准确率很高,对犬小孢子菌（75%）、镰刀菌属（50%）、新月弯孢霉（46.2%）准确率较低,对丝状真菌鉴定的总体准确率为86.36%,与ITS测序分析符合率为83.97%。     

Sites of interaction of a precursor polypeptide on the export chaperone SecB mapped by site-directed spin labeling

Export of protein into the periplasm of Escherichia coli via the general secretory system requires that the transported polypeptides be devoid of stably folded tertiary structure. Capture of the precursor polypeptides before they fold is achieved by the promiscuous binding to the chaperone SecB. SecB delivers its ligand to export sites through its specific binding to SecA, a peripheral component of the membrane translocon. At the translocon the ligand is passed from SecB to SecA and subsequently through the SecYEG channel. We have previously used site-directed spin labeling and electron paramagnetic resonance spectroscopy to establish a docking model between SecB and SecA. Here we report use of the same strategy to map the pathway of a physiologic ligand, the unfolded form of precursor galactose-binding protein, on SecB. Our set of SecB variants each containing a single cysteine, which was used in the previous study, has been expanded to 48 residues, which cover 49% of the surface of SecB. The residues on SecB involved in contacts were identified as those that, upon addition of the unfolded polypeptide ligand, showed changes in spectral line shape consistent with restricted motion of the nitroxide. We conclude that the bound precursor makes contact with a large portion of the surface of the small chaperone. The sites on SecB that interact with the ligand are compared with the previously identified sites that interact with SecA and a model for transfer of the ligand is discussed.