A novel approach for the determination of freshness and identity of trouts by MALDI-TOF mass spectrometry

Properly handled fish is usually marketed as “fresh fish” until day 10 after fishing. About 40% of the total fishery that is used for direct human consumption is marketed in fresh form stored at temperatures up to +2 °C. Currently, there are no validated methods available for controlling the recommended period of storage. Apart from being a potential source for food fraud, spoiled fish represents a major source of foodborne illnesses and intoxications.In this study, a rapid MALDI-TOF mass spectrometry based screening method was developed using the vitreous fluid of fish eyes as specimen for the examination of different days of storage. The vitreous fluid was collected from n = 100 freshly fished brown trouts at day 0, 3, 7, 9, and 11 post mortem (n = 20 brown trouts each day of examination). The samples were immediately measured by MALDI-TOF mass spectrometry in linear positive mode (mass range m/z 2000–20,000 Da). For quality assurance the experiment was repeated with a set of brown trouts (n = 100) originating from the same fish farm and with brown trouts (n = 100) originating from a different fish farm. For specificity testing rainbow trouts (n = 10) were examined accordingly. All obtained mass spectra were processed by means of MALDI Biotyper OC 3.1 and ClinProTools 3.0 software.The MALDI Biotyper approach showed limited applicability for the identification of the time of storage. However, it was suitable to reliably discriminate between the closely related species brown and rainbow trout. Processing by ClinProTools revealed four crucial mass peaks (m/z 2594 Da, m/z 4857 Da, m/z 4879 Da, m/z 4899 Da) which enabled a reliable differentiation between day 0 and 3, 7, 9, 11 (rate of correct identification > 90%) as well as the differentiation between day 3 and 7, 9, 11 (rate of correct identification > 72%). However, this approach showed limited applicability within the end of the tested period of storage when comparing between day 7, 9, or 11.     

Presence of Bacillus species in pasteurised milk and their phenotypic and genotypic antimicrobial resistance profile

High-temperature short-time (HTST) pasteurisation is not sufficient for complete elimination of Bacillus sp. in milk. Hence, it is important to assess their prevalence, phenotypic and genetic antimicrobial resistance profiles in pasteurised milk. In this study, we investigated Bacillus prevalence and their phenotypic resistance to 25 antimicrobials and prevalence of five plasmid antimicrobial resistance genes. All isolates were resistant to β-lactams; most isolates were susceptible to chloramphenicol (n = 52), ciprofloxacin, meropenem, sulphonamides, tetracycline and vancomycin (n = 56). Intriguingly, despite phenotypic sulphonamide and tetracycline sensitivity, Sul2 and tetA genes were detected in different Bacillus species, signifying a potential risk of horizontal gene transfer of these mobile resistance genes through pasteurised milk.     

利用质谱分析法从酶谱鉴定嗜热真菌木聚糖酶

通过对前期筛选到的一株嗜热真菌Talaromyces thermophilus WYN9进行产木聚糖酶的诱导和非诱导培养。诱导培养的上清液中可以检测到木聚糖酶酶活。在非变性胶和变性胶SDS-PAGE中,诱导和非诱导的酶谱条带差异明显。以非诱导酶谱为对照,将诱导酶谱中与其有显著差异的条带切下,回收蛋白,测定酶活。将有木聚糖酶酶活的蛋白通过SDS-PAGE进一步分离纯化,切胶后使用胶内酶切法进行预处理,使用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）进行分析。结果表明该蛋白与拟青霉属Paecilomyces sp. J18的内切木聚糖酶高度同源。根据质谱分析结果设计兼并引物,通过交错式热不对称PCR（thermal asymmetric interlaced PCR,TAIL-PCR）扩增,获得了TtXynA基因全长。根据Kimura双参数模型用临近相邻法构建了TtXynA及其同源蛋白的进化树。     

Amino acid composition,antinutrients and allergens in the peanut protein fraction obtained by an aqueous enzymatic process

Enzyme-assisted aqueous extraction (EAE) of peanut kernel was used to extract oil and protein. The aqueous fraction (AF) obtained by EAE had a better essential amino acid profile than the residues obtained by solvent extraction (Rs) and cold pressing (Rc). No major difference in the trypsin inhibitor activity among AF, Rs and Rc was observed; however, the trypsin inhibitor activity was drastically reduced in the residue obtained after EAE. AF was subjected to MALDI-TOF/MS, revealing it to be rich in peptides (107) with molecular masses from m/z 700 to 2369 Da. AF had an extremely low phytate content and was rich in peptides, which could be used as a food supplement. ESI-MS/MS data were used for the identification of major peanut allergens, viz., Ara h1, h3, h6–8. Their allergenic potential needs to be established.     

Deconvolution of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry isotope patterns to determine ratios of A-type to B-type interflavan bonds in cranberry proanthocyanidins

A method to deconvolute overlapping isotope patterns in positive mode matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was developed to determine ratios of A- to B-type interflavan bonds in proanthocyanidins that were isolated from cranberry (Vaccinium macrocarpon, Ait.) press cake (c-PAC). Precision and accuracy was validated for binary mixtures of procyanidins A2 and B2. Deconvolution of c-PAC spectra indicated that oligomers with one or more A-type interflavan bonds occur in a higher proportion than oligomers with all B-type interflavan bonds. c-PAC with at least one A-type bond accounted for more than 91% of the oligomers between trimers and undecamers. The c-PAC isotope patterns are highly repeatable, suggesting that the method can be applied to authentication, standardization and efficacy of cranberry products in relationship to urinary tract health. This is the first time MALDI-TOF MS has been used for estimating ratios of A- to B-type bonds in PAC.     

不同品种茶叶单宁的含量与结构测定

测定了本山、黄旦和铁观音三个品种茶树成熟叶片的总酚及可溶缩合单宁含量,并通过基质辅助激光解吸附飞行时间质谱(MALDI-TOF MS)研究了茶叶缩合单宁的基本结构。结果显示,三种茶叶均具有较高的总酚含量,大约为200mg/g;三种茶叶的缩合单宁是以(表)儿茶素(EC/C)和(表)棓儿茶素(EGC/GC)为基本结构单元的均聚物和杂聚物,且大部分聚合物的结构单元之间存在A 型和B 型两种连接方式,其中本山和黄旦缩合单宁的最高聚合度要大于铁观音。     

基质辅助激光解析电离飞行时间质谱法提取阳澄湖全谱图范围内大闸蟹蛋白条件优化

目的 建立最佳的蟹肉组织蛋白分析方案,获得最佳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)图,为基于MALDI-TOF MS技术的阳澄湖大闸蟹产地鉴别...     

含二硫键的蛋白质/多肽的MALDI-TOF MS源内裂解研究

应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)法研究含二硫键的人胰岛素与甘精胰岛素酶解液的源内裂解(ISD)。比较了不同基质种类及不同结晶状态对含二硫键的人胰岛素与甘精胰岛素酶解液的源内裂解的影响。结果表明,含二硫键的蛋白质的ISD发生受激光点照射位置的影响,在不同基质与结晶形态的条件下,含二硫键的蛋白质的ISD碎片信息不同。通过分析比较,含二硫键的蛋白质的ISD较容易控制,并且其基质的种类及结晶状态作用很关键。需要获得大量碎片时,使激光照射在样品和阿魏酸(FA)基质形成的大结晶处;不希望出现碎片时,可使用2,4,6-三羟基苯乙酮(THAP)为基质,或使激光照射在样品和其他基质形成的细小均匀结晶处。     

稀土金属元素标记行为考察

稀土金属元素因其与双功能试剂的螯合效率高、对肽段的反相色谱行为和质谱的离子化效率影响小、检测背景低,以及过量的金属离子在检测时不会产生干扰等优点,近年来在生物标记结合质谱定量分析研究领域引起了关注。选取稀土金属中单同位素铽（Tb）、钇（Y）、铥（Tm）、钬（Ho）和镥（Lu）5种元素,通过双功能试剂二乙三胺五乙酸双酸酐（DTPA双酸酐）对合成肽段及蛋白质酶切产物标记的过程进行研究,包括对标记条件进行优化,对该质量标签的色谱行为和共洗脱、标记稳定性、离子交换、动态范围、质谱检测限等现象进行考察。结果表明：铥和钬两元素结合效率最高,分别达到了92.5%和89.9%;稳定性考察结果表明,质量标签在4 ℃下放置一周仍可检测到95%,若将标记好的标签用于定量研究,则放置时间不能超过两周;多金属标记质量标签可以共洗脱;两种金属浓度差不超过6倍时,由离子抑制带来的影响较小;离子交换结果表明,只有很少部分发生了离子交换,对标记实验没有影响。