杂交兰花色花香生物合成途径的转录组分析

该研究以杂交兰(Cymbidium hybrid)不同花色花香品种‘玉凤’(K18,黄色)和‘福韵丹霞’(K24,紫红色)为材料,采用RNA-Seq技术获得杂交兰不同花期的花朵转录组数据,分析杂交兰不同时期花色/花香相关基因的表达变化,探讨杂交兰花色花香形成的分子机理,为杂交的定向改良和新品种选育提供依据。结果表明:(1)K18和K24分别获得11914和6793个差异表达基因;KEGG注释显示,有58个差异基因与花色花香合成相关;进一步分析发现,类黄酮是K18和K24的主要花色素,其合成基因在小花蕾期显著上调,其中K18通过查尔酮异构酶基因(CHI)、类黄酮-3′,5′羟化酶基因(F3′5′H)和黄酮醇合酶基因(FLS)途径生成黄酮醇,K24则通过花青素合成酶基因(ANS)途径生成花青素。(2)qRT-PCR验证表明,萜类骨架基因羟甲基戊二酰辅酶基因(HMGS)、羟基-3-甲基戊二酰辅酶A还原酶基因(HMGR2)和甲羟戊酸-5-焦磷酸合酶基因(MVD)等的表达量均在K18盛花期最高,同时6个下游的萜烯合酶(TPS)基因在K18中表达量比K24上调100倍以上。(3)定量分析表明,K24、K18中的花色素苷总含量分别为608.74、122.28μg·g-1,K18花色苷含量仅为K24的20%;K24花中色素物质主要成分为矢车菊素苷,使其花色呈红色;K18中飞燕草素苷含量相对较高(花色为黄色)。研究推测,ANS基因的较高表达可能是K24花瓣中花色苷含量高于K18的原因之一,K18通过CHI、F3′5′H、FLS途径积累了大量黄酮醇而不是花青素,从而影响了花朵颜色的决定。     

杂交兰及其花艺突变体中类黄酮成分差异的代谢-转录组联合分析

为探索杂交兰花艺突变体变异的分子机理,该研究以杂交兰‘玉凤’及其花艺突变体‘双艺金龙’为材料,利用靶向代谢组学和转录组学鉴定两者中类黄酮化合物含量差异及其相关通路上的差异表达基因。结果表明：(1)代谢组分析发现,杂交兰‘玉凤’和‘双艺金龙’花瓣中共检测到271种类黄酮代谢物,其中黄酮醇类和黄酮类代谢物的相对含量约占总类黄酮的30%～50%;共检测到38个差异代谢物(15个上调,23个下调);‘玉凤’中差异最高的代谢物二氢山奈酚-7-O-葡萄糖苷(黄酮醇类)含量是‘双艺金龙’的124 444倍,‘双艺金龙’中差异最高的代谢物3,5,7,3′,4′-五羟基-5′-异戊二烯基黄酮(黄酮醇类)含量是‘玉凤’的7 244倍。(2)KEGG分析显示,差异代谢物显著富集在类黄酮、黄酮和黄酮醇生物合成途径。其中,在类黄酮生物合成途径上,根皮苷、黄腐醇、二氢山奈酚、二氢杨梅素和表没食子儿茶素含量升高,柚皮苷和二氢槲皮素含量降低;在黄酮和黄酮醇生物合成途径上,三叶豆苷和槲皮素含量均下降。(3)转录组分析共筛选到563个差异表达基因,与‘玉凤’相比,‘双艺金龙’中有220(39.1%)个基因上调表达,343...     

小苍兰花色色素成分及稳定性分析

以小苍兰(Freesia refracta)16个不同花色品种及后代为试验材料,对花瓣色素用特征显色反应和紫外-可见光谱扫描,分析其色素的成分和花色素苷的稳定性.结果表明,小苍兰花色的色素属于类黄酮化合物,含黄酮和花色素苷类物质,可能含有异黄酮,不含黄酮醇、二氢黄酮、二氢黄酮醇、查耳酮和橙酮,其中黄色系品种及后代还含有类胡萝卜素.避光下小苍兰花色素苷的稳定性要强于光照;温度对花瓣色素的稳定性有一定的影响.     

香雪兰花瓣的花色苷组成

为研究不同品种香雪兰的花色苷组成、含量及与花色表型之间的关系,阐明香雪兰花色形成机理,该研究以不同花色的香雪兰(Freesia hybrida) 11个品种为材料,采用英国皇家园艺学会比色卡(RHSCC)和色差仪进行花色描述,利用特征颜色反应初步确定色素类型,通过pH示差法测定花瓣中总花色苷的含量,进而利用UPLC-Q-TOF-MS技术分析各品种花瓣中花色苷种类和相对含量。结果表明:11个所选品种涵盖香雪兰四大色系,即白色系、黄色系、红色系、蓝紫色系;所选品种都含有黄酮类化合物,不含或含有极低量的类胡萝卜素,除‘White River’‘Fragrant Sunburst’‘Gold River’‘Tweety’外,均含有花色苷;‘Red Passion’花瓣中总花色苷含量最高,最低是‘Lovely Lavender’,其含量仅为‘Red Passion’的24%;在香雪兰花瓣中共检测出10个花色苷组分,分别为飞燕草-二葡萄糖苷、矢车菊素-二葡萄糖苷、矮牵牛素-二葡萄糖苷、飞燕草素-3-O-葡萄糖苷、矢车菊素-3-O-葡萄糖苷、芍药素-二葡萄糖苷、锦葵素-二葡萄糖苷、矮牵牛素-3-O-葡萄糖苷、芍药素-3-O-葡萄糖苷、锦葵素-3-O-葡萄糖苷;红色系品种‘Red Passion’和‘上农红台阁’花瓣中主要成分为矢车菊素类化合物,蓝紫色系品种‘Pink Passion’‘Castor’‘上农淡雪青’和‘上农紫玫瑰’花瓣中主要成分为矮牵牛素类和锦葵素类化合物,‘Lovely Lavender’花瓣仅含飞燕草素类化合物。研究表明不同品种香雪兰花瓣颜色的呈现与花色苷种类有关,花瓣着色程度则与花瓣中花色苷总含量成正比。该研究结果为新品种培育、花色改良和育种工作提供理论依据。     

不同红叶性状桃叶片花色素苷种类鉴定及呈色规律研究

以早熟桃(Prunus persica,‘早美’、‘春蕾’)和红叶桃(Prunus persica f.atropur purea,‘筑波5号’、‘洛格红叶’)两种不同叶片呈色类型桃品种为试材,在5~9月份对其呈色过程中叶片色泽、花色素组成进行了测定,并对其叶片色差值与色素组成间的相关性进行了分析,探讨早熟桃和红叶桃叶片呈色差异的生理机制。结果表明:(1)红叶桃叶片5月下旬叶片出现"返青"现象,叶片a*值由正值变为负值;相反早熟桃叶片6月份果实采收后叶片由鲜艳绿色逐渐变为红紫色,叶片a*值逐渐增大。(2)在试验所选的4个品种中共发现5类花色素苷,早熟桃含有3类,红叶桃含有4类,矢车菊素-3-葡萄糖苷和矢车菊素-3-芸香糖苷为两种类型桃叶片所共有的且含量相对较高的花色素苷;呈色过程中叶片花色素苷种类基本不变,但各色素含量发生了明显的变化。(3)多元回归分析显示:红叶桃叶片与早熟桃叶片的a*值分别与矢车菊素-3-葡萄糖苷、矢车菊素-3-芸香糖苷含量呈正相关关系,且均与Ant/Chl呈负相关关系。研究表明,矢车菊素为桃叶片呈现红色的物质基础,两种类型桃叶片呈色差异并非所含花色素苷种类不同所致,而与不同种类花色素苷含量有关。     

铁筷子花瓣中花青素苷成分及含量对其呈色的影响北大核心CSCD

该研究以7个品种铁筷子(Helleborus thibetanus Franch.)为试验材料,借助目视测色、RHSCC比色卡、色差仪进行花色表型的测定,采用高效液相色谱法-光电二极管阵列检测方法(HPLC-DAD)及高效液相色谱-电喷雾离子化-质谱联用技术(HPLC-ESI-MS)测定分析铁筷子花瓣中花青素苷成分及含量,以探究不同品种铁筷子的花色与花青素苷成分及含量之间的关系。结果显示:(1)紫色系品种花瓣的a*值最高b*值最低,黄色系品种花瓣的b*值最高a*值最低,不同品种的铁筷子花色越深L*值越低。(2)从5个有花青素苷积累的铁筷子品种中检测出11种花青素苷成分,分别为6种矢车菊素苷,4种飞燕草素苷,1种矮牵牛素苷;供试的铁筷子材料中红色系2个品种的花青素苷含量最高,紫色系品种次之;矢车菊素苷与飞燕草素苷为影响铁筷子花瓣呈色的主要色素物质。(3)不同种类的花青素和修饰基团的差异,导致铁筷子花瓣呈现不同的色彩,含有多种酰基化修饰的飞燕草素苷使铁筷子花色蓝移进而使花色加深。(4)相关分析表明,铁筷子花瓣的L*值与a*值呈显著负相关关系,与b*值呈显著的正相关关系;L*值与总花青素苷含量呈显著负相关关系,且随着花青素苷含量的累积a*值增加,花色红移。研究表明,花青素苷的成分及含量是导致铁筷子花瓣呈现不同颜色的主要原因,矢车菊素苷和飞燕草素苷的互作以及酰基化的修饰使铁筷子呈现不同程度的紫色,花青素苷的不同累积量影响了花瓣颜色的明暗变化,从而使铁筷子花瓣颜色丰富。     

不同花色菊花品种花色素结构基因的表达特性

对红色、黄色、粉紫色和白色菊花品种不同开放度的花序舌状花中CHS、CHI、DFR、F3H、F3′H和3GT基因的表达量进行了相对定量分析。结果表显示:6个基因的表达因不同花色、不同发育阶段而异。‘钟山红鹰’(红色)中各基因的表达量均较高,且均在Ⅱ(松蕾期)或Ⅲ(半开期)期达到峰值,其中DFR、3GT基因的表达量远高于其他花色品种。‘金陵娇黄’(黄色)中CHS、CHI基因表达量较高,且Ⅰ(紧蕾期)、Ⅱ期表达量高于Ⅲ、Ⅳ(盛开期)期;3GT、DFR基因表达量分别高或低于‘金陵笑靥’(粉紫色)品种中相应基因的表达量,但均比红色品种低;F3H在4个品种中表达量最低,F3′H表达量接近或略低于红色或粉紫色品种,且各阶段表达水平较稳定。‘金陵笑靥’中DFR表达量仅次于‘钟山红鹰’,3GT和CHS表达量低于红色与黄色品种。‘钟山雪桂’(白色)中各基因仅有微量表达,除F3H外各基因的表达量明显低于其他花色品种。研究表明,花色素结构基因DFR、3GT是菊花花色素合成的关键基因,DFR很可能是限速关键基因,一定表达水平的CHS、CHI也是菊花花色素合成所必须的,F3H基因与花色素合成不存在直接相关。     

欧洲卫矛秋冬转色期叶色变化的生理机制

以3个欧洲卫矛(Euonymus europaea)品种‘矮生’(Euonymus europaea‘Pumilis’)、‘八仙花’(E.europaea‘Aldenhamensis’)和‘白果’(E.europaea‘Albus’)为试验材料,测定其秋冬转色期叶色参数及各项生理指标的变化,分析色素与叶色参数、可溶性糖、相关酶的关系,探讨欧洲卫矛转色期叶片的呈色机理,为后期园林应用提供依据。结果表明:(1)‘八仙花’、‘白果’叶片明度参数L*值、色素参数b*值(黄/蓝)呈先升后降的单峰曲线,‘矮生’的L*值、b*值呈先降后升的单峰曲线,3个品种色素参数a*值(红/绿)在转色期间均不断上升。(2)各品种叶片叶绿素含量随生育期整体呈下降的趋势,主要是叶绿素a减少,叶绿素b与类胡萝素下降较为平稳;花色素苷含量、花色素苷/叶绿素比值呈上升趋势,类胡萝素/叶绿素比值整体呈下降的趋势。(3)各品种叶片可溶性糖含量随生育期呈先升后降的单峰曲线,它们的PAL活性呈波动上升趋势,POD活性整体呈下降趋势,PPO活性呈缓慢上升趋势。(4)相关性分析显示,3个品种叶色参数a*值与花色素苷含量均呈极显著正相关关系,‘矮生’花色素苷含量与叶绿素、类胡萝素含量呈显著负相关关系,‘八仙花’、‘白果花’的花色素苷含量与叶绿素、类胡萝卜素含量呈不显著负相关关系。研究发现,影响欧洲卫矛秋冬转色期叶片变红的主要因素是叶绿素、花色素苷和可溶性糖相对含量及PAL、PPO活性,类胡萝卜素含量对叶片变红的贡献不大,POD活性对叶片变红无直接影响。     

滇山茶狮子头及其芽变品种大玛瑙间花色差异分析

滇山茶是世界著名观赏花木,花色是其重要的观赏性状。滇山茶狮子头花色为深红色,而其芽变品种大玛瑙是滇山茶中唯一红白双色的名贵品种,极具观赏价值。以上述2个品种为研究材料,采用RHSCC比色卡比色法和色差仪测定2个品种滇山茶花蕾期和盛花期花瓣的花色表型,并基于转录组与代谢组分析挖掘呈色相关的关键代谢物及关键基因。花青素靶向代谢分析表明,在滇山茶2个品种中共鉴定出28种花青素代谢物,其中狮子头与大玛瑙红色区域花瓣间没有差异代谢物,狮子头与大玛瑙白色区域花瓣间的关键差异代谢物为矢车菊素-3-O-桑布双糖苷、原花青素B2、原花青素B3、阿福豆苷,大玛瑙花瓣的红白区域关键差异代谢物为矢车菊素-3-O-桑布双糖苷、原花青素B2、阿福豆苷。转录组KEGG分析结果表明,苯丙醇生物合成和类黄酮生物合成途径与大玛瑙红白双色花瓣的形成有关;植物激素信号转导和昼夜节律-植物途径与滇山茶花色芽变有关。转录代谢联合分析共筛选出与滇山茶呈色高度相关的差异表达基因共17条,包括4条CHS、3条HCT、2条F3′H、1条LAR、5条MYB和2条b HLH。本研究结果对进一步揭示花色芽变育种具有一定的参考意义。     

山樱花品种间花色差异的代谢组学研究

山樱花是世界著名的观花类植物，花色是其最重要的观赏特征。为探究影响山樱花品种间花色差异的代谢通路及关键代谢产物变化，该文利用LC-MS/MS技术对白色、绿色和粉色的山樱花品种进行花青素靶向代谢组学比较分析。结果表明：(1)共检测到42种花青素物质，主要包含矮牵牛素、飞燕草素、黄酮类化合物、锦葵色素、芍药花素、矢车菊素、天竺葵素和原花青素8种物质。(2)差异代谢花青素25种，包括11种下调、14种上调，其中有7种花青素在粉色花瓣中显著富集。(3)KEGG通路注释发现差异代谢物在花青素生物合成通路中显著富集，结合聚类结果发现矮牵牛素-3-O-葡萄糖苷是山樱花品种间花色差异产生的关键代谢物。该研究揭示了山樱花花色差异的代谢机理，为后续山樱花花色分子调控机制研究提供了一定的理论依据，也为新品种花色改良和选育提供了一定的科学参考。     

Metabolite profiling of saponins in <Emphasis Type="Italic">Balanites</Emphasis><Emphasis Type="Italic">aegyptiaca</Emphasis> plant tissues using LC (RI)-ESI/MS and MALDI-TOF/MS

Saponins are a major family of secondary metabolites which consist of a sugar moiety glycosidically linked to a hydrophobic aglycone (sapogenin). In recent years the interest in saponins has increased significantly because of their diverse properties as natural detergents and foaming agents, their cardiac, immunostimulating, and anti-cancer activity, as well as other health promoting functions. This study deals with metabolitic analysis of saponins from methanolic extracts of fruit mesocarp (ME), seed kernel (KE) and root (RE) of Balanites aegyptiaca (L.) Del. (desert date) plant grown in Israel using LC (RI)-ESI/MS and MALDI-TOF/MS. The structural assignment was carried out by fragmentation experiments of LC (RI)-ESI/MS and literature data. The study has revealed that, all together, twenty-four furostanol saponins were found in ME, KE and RE. Of these, four saponins are found only in ME, five only in KE and six only in RE. Diosgenin was found to be the sole aglycone in all the saponins. The smallest saponin (MW 740 Da) was found with two sugar units (glucose) and the largest saponin (MW 1678 Da) was found with eight sugar units (5 glucose, 2 rhamnose and 1 xylose) attached to diosgenin. The results suggest that MALDI-TOF/MS with positive ion mode is particularly effective for determining the metabolites of saponins in B. aegyptiaca plant tissues. MALDI-TOF/MS not only verified the results of the LC (RI)-ESI/MS, but also identified additional saponins that are now systematically organized in a database of B. aegyptiaca saponins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Induced proteome of Trichoderma harzianum by Botrytis cinerea

As a notable biocontrol agent, Trichoderma harzianum can antagonize a diverse array of phytopathogenic fungi, including Botrytis cinerea, Rhizoctonia solani and Fusarium oxysporum. Elucidating the biocontrol mechanism of T. harzianum in response to the pathogens enables it to be exploited in the control of plant diseases. Two-dimensional gel electrophoresis (2-DE) was performed to obtain secreted protein patterns of T. harzianum ETS 323, grown in media that contained glucose, a mixture of glucose and deactivated B. cinerea mycelia, deactivated B. cinerea mycelia or deactivated T. harzianum mycelia. Selected protein spots were identified using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Ninety one out of 100 excised protein spots were analyzed and some proteins were sequence identified. Of these, one l-amino acid oxidase (LAAO) and two endochitinases were uniquely induced in the media that contained deactivated B. cinerea mycelia as the sole carbon source. Activities of the cell wall-degrading enzymes (CWDEs), including β-1,3-glucanases, β-1,6-glucanases, chitinases, proteases and xylanases, were significantly higher in media with deactivated B. cinerea mycelia than in other media. This finding suggests that the cell wall of B. cinerea is indeed the primary target of T. harzianum ETS 323 in the biocontrol mechanism. The possible roles of LAAO and xylanase were also discussed.     

Evaluation of Chemical Derivatisation Methods for Protein Identification using MALDI MS/MS

In proteomic studies, assigning protein identity from organisms whose genomes are yet to be completely sequenced remains a challenging task. For these organisms, protein identification is typically based on cross species matching of amino acid sequence obtained from collision induced dissociation (CID) of peptides using mass spectrometry. The most direct approach of de novo sequencing is slow and often difficult, due to the complexity of the resultant CID spectra. For MALDI-MS, this problem has been addressed by using chemical derivatisation to direct peptide fragmentation, thereby simplifying CID spectra and facilitating de novo interpretation. In this study, milk whey proteins from the tammar wallaby (Macropus eugenii) were used to evaluate three chemical derivatisation methods compatible with MALDI MS/MS. These methods included (i) guanidination and sulfonation using chemically-assisted fragmentation (CAF), (ii) guanidination and sulfonation using 4-sulfophenyl isothiocyanate (SPITC) and (iii) derivatising the epsilon-amino group of lysine residues with Lys Tag 4H. Derivatisation with CAF and SPITC resulted in more protein identification than Lys Tag 4H. Sulfonation using SPITC was the preferred method due to the low cost per experiment, the reactivity with both lysine and arginine terminated peptides and the resultant simplified MS/MS spectra.*Australian Peptide Conference Issue.**This project was funded by an ARC Linkage grant to Deane supported by TGR Biosciences and facilitated by access to the Australian Proteome Analysis Facility established under the Australian Government’s Major National Research Facilities program.     

Single-cell proteomic analysis of glucosinolate-rich S-cells in Arabidopsis thaliana

Single-cell analysis is essential for understanding the processes of cell differentiation and metabolic specialisation in rare cell types. The amount of single proteins in single cells can be as low as one copy per cell and is for most proteins in the attomole range or below; usually considered as insufficient for proteomic analysis. The development of modern mass spectrometers possessing increased sensitivity and mass accuracy in combination with nano-LC–MS/MS now enables the analysis of single-cell contents. In Arabidopsis thaliana, we have successfully identified nine unique proteins in a single-cell sample and 56 proteins from a pool of 15 single-cell samples from glucosinolate-rich S-cells by nanoLC–MS/MS proteomic analysis, thus establishing the proof-of-concept for true single-cell proteomic analysis. Dehydrin (ERD14_ARATH), two myrosinases (BGL37_ARATH and BGL38_ARATH), annexin (ANXD1_ARATH), vegetative storage proteins (VSP1_ARATH and VSP2_ARATH) and four proteins belonging to the S-adenosyl-l-methionine cycle (METE_ARATH, SAHH1_ARATH, METK4_ARATH and METK1/3_ARATH) with associated adenosine kinase (ADK1_ARATH), were amongst the proteins identified in these single-S-cell samples. Comparison of the functional groups of proteins identified in S-cells with epidermal/cortical cells and whole tissue provided a unique insight into the metabolism of S-cells. We conclude that S-cells are metabolically active and contain the machinery for de novo biosynthesis of methionine, a precursor for the most abundant glucosinolate glucoraphanine in these cells. Moreover, since abundant TGG2 and TGG1 peptides were consistently found in single-S-cell samples, previously shown to have high amounts of glucosinolates, we suggest that both myrosinases and glucosinolates can be localised in the same cells, but in separate subcellular compartments. The complex membrane structure of S-cells was reflected by the presence of a number of proteins involved in membrane maintenance and cellular organisation.     

Cloning, expression, and enzymatic activity of Acinetobacter baumannii and Klebsiella pneumoniae acetyl-coenzyme A carboxylases

Pathogenic Gram-negative bacteria are a major public health concern because they are causative agents of life-threatening hospital-acquired infections. Due to the increasing rates of resistance to available antibiotics, there is an urgent need to develop new drugs. Acetyl-coenzyme A carboxylase (ACCase) is a promising target for the development of novel antibiotics. We describe here the expression, purification, and enzymatic activity of recombinant ACCases from two clinically relevant Gram-negative pathogens, Acinetobacter baumannii and Klebsiella pneumoniae. Recombinant ACCase subunits (AccAD, AccB, and AccC) were expressed and purified, and the holoenzymes were reconstituted. ACCase enzyme activity was monitored by direct detection of malonyl-coenzyme A (malonyl-CoA) formation by liquid chromatography tandem mass spectrometry (LC–MS/MS). Steady-state kinetics experiments showed similar k_cat and K_M values for both enzymes. In addition, similar IC₅₀ values were observed for inhibition of both enzymes by a previously reported ACCase inhibitor. To provide a higher throughput assay suitable for inhibitor screening, we developed and validated a luminescence-based ACCase assay that monitors ATP depletion. Finally, we established an enzyme activity assay for the isolated AccAD (carboxyltransferase) subunit, which is useful for determining whether novel ACCase inhibitors inhibit the biotin carboxylase or carboxyltransferase site of ACCase. The methods described here could be applied toward the identification and characterization of novel inhibitors.     

Toxin profile of Alexandrium catenella from the Chilean coast as determined by liquid chromatography with fluorescence detection and liquid chromatography coupled with tandem mass spectrometry

The profile of tetrahydropurine neurotoxins associated with paralytic shellfish poisoning (PSP) was determined from a Chilean strain of the marine dinoflagellate Alexandrium catenella. The toxin composition was compared with that of toxic shellfish, presumably contaminated by natural blooms of A. catenella from the same region in southern Chile. Ion pair-liquid chromatography with post-column derivatization and fluorescence detection (LC-FD) was employed for relative quantitative analysis of the toxin components, whereas unambiguous identification of the toxins was confirmed by tandem mass spectrometry (LC–MS/MS). In the dinoflagellate strain from Chile, the N-sulfocarbamoyl derivatives (C1/C2, B1) and the carbamoyl gonyautoxins GTX1/GTX4 comprise >90% of the total PSP toxin content on a molar basis. This toxin composition is consistent with that determined for A. catenella populations from the Pacific coast in the northern hemisphere. The characteristic toxin profile is also reflected in the shellfish, but with evidence of epimerization and metabolic transformations of C1 and C2 to GTX2 and GTX3, respectively. This work represents the first unequivocal identification and confirmation of such PSP toxin components from the Chilean coast.     

Chemical Modification of Hamster Arylamine N-Acetyltransferase 2 with Isozyme-Selective and Nonselective N-Arylbromoacetamido Reagents

Arylamine N-acetyltransferases (NATs) catalyze a variety of biotransformation reactions, including N-acetylation of arylamines and O-acetylation of arylhydroxylamines. Chemical modification of hamster recombinant NAT2 with 2-(bromoacetylamino)fluorene (Br-AAF) and bromoacetanilide revealed that Br-AAF is an affinity label for the enzyme whereas bromoacetanilide inactivates NAT2 through a bimolecular alkylation process. Electrospray ionization quadrupole time-of-flight mass spectrometry analysis of Br-AAF-treated NAT2 showed that a single molecule of 2-acetylaminofluorene had been adducted. Peptide sequencing with tandem mass spectrometry identified the catalytically essential Cys68 as the alkylated amino acid. Br-AAF exhibits similar affinity for hamster NAT1 and NAT2, but is a more effective inactivator of NAT1 because, subsequent to the formation of a reversible enzyme-Br-AAF complex, the rate of alkylation of NAT1 is greater than the rate of alkylation of NAT2. Bromoacetanilide alkylates Cys68 and, to a lesser extent, Cys237 of NAT2; it does not exhibit significant selectivity for either NAT1 or NAT2.