This research was generously funded by the NIH NCI IMAT Program Grant R33 (CA147988-02), the NIH NIGMS Graduate Training in Molecular Biotechnology at NC State Grant (T32GM008776), the US Dept. of Education GAANN Fellowship Program in Molecular Biotechnology at NC State Grant (P200A140020), the W.M. Keck Foundation, and North Carolina State University. Hen plasma was obtained with the assistance of Dr. James N. Petitte and Rebecca Wysocky in the NC State University Dept. of Poultry Science.

1.蛋白质和糖蛋白变性及烷基化<ol><li>对于标准制剂，负载2.5微升的糖蛋白（ 例如 ，胎球蛋白或RNA酶B）的0.1微克/微升溶液到30 kDa的或10kDa的分子量（MW）截止滤波器。 <ol><li>生物血浆样品，通过标准布拉德福德19或二辛可宁酸20（BCA）蛋白质测定法检查蛋白质浓度。在pH〜7至10-100毫克/毫升的总蛋白之间包含稀的等离子体，如果需要的话，用100mM碳酸氢铵（NH 4 HCO 3）的H 2 O（PNG酶消化缓冲液）。负载2.5微升血浆（25-250微克蛋白质）上的过滤器。 注意:此协议只被从在乙二胺四乙酸（EDTA）的存在下收集血液样品的血浆进行试验。 </li></ol></li><li>添加2微升的1M二硫苏糖醇溶液（DTT）至在过滤器中的每个样本。 </li><li>用稀释20样本0微升PNG酶消化缓冲。 </li><li>盖上过滤器样品管，轻轻涡旋，小心不要打扰其座位过滤器。在56℃孵育样品30分钟以变性蛋白和糖蛋白。 </li><li>烷基化的样品中，添加50微升的1M碘乙酰胺，得到〜200mM的终浓度，并在37°C孵育60分钟。 注意:如果蛋白质组学分析是不希望，步骤1.5可以跳过。 </li><li>通过以14,000 xg离心离心样品40分钟浓缩变性的糖蛋白在过滤器上。丢弃流过。 </li></ol> 4. N -连接聚糖酶消化<ol><li>用100微升洗涤样品PNG酶消化缓冲。集中在过滤器上的糖蛋白在14,000rpm xg离心20分钟，丢弃流过。 <ol><li>重复洗涤和浓缩步骤两次，总共三次，得到的过滤器的死体积（〜5微升）的浓缩物。抛弃一切流过。 </li><li>丢弃收集瓶洗一次完成。收集在一个新的收集瓶今后所有的洗脱液和洗涤。 注:PNG酶消化缓冲的H 2 18 O可能在酶F消化步骤为去糖基化位点的天冬酰胺，这成为化学脱酰胺稳定同位素标记使用。 </li></ol></li><li>添加2微升的游离甘油PNG酶F（75000点¯x单位/ ml）转移到新鲜的收集瓶中后进行过滤。加入98微升PNG酶消化的缓冲，使总体积为100微升，轻轻移液器上下在过滤器混合。酶促消化在步骤2.2.1，2.2.2，OR 2.2.3条件下的聚糖。 注:可用于蛋白质的去糖基化的三种方法。全权糖组学分析（无蛋白质组学），建议在步骤2.2.1潜伏期长。对于糖组学和蛋白质组学研究，步骤2.2.2和2.2.3将生产高品质的肽用最少的非SPecific脱酰胺。 <ol><li>只有糖组学消化协议（18小时）:在37℃孵育样品18小时酶促裂解所有N -glycans。 </li><li>尖峰在消化协议（SPI）:在50℃下孵育样品2小时。工作迅速，除去额外的2微升无甘油的PNGase F（75000点¯x单位/毫升）的样品和尖峰。轻轻涡样品1-2秒混合。孵育在50℃下将样品另外2小时。 </li><li>微波消解协议（MD）:在离心管浮架放置样品。在一个1升烧杯浮子样品填充有1升的去离子（DI）水。放置烧杯在微波炉（950瓦）与旋转板的中心。微波中在60％的功率（570瓦）5分钟。冷却，除去样品并在室温下保持2分钟。然后微波在60％的电力额外的5分钟。 注:微波功率设置将需要根据微波的最大输出功率进行调整。平均功率要追究模型之间不变。 </li></ol></li></ol> 3. 氮洗脱-glycans <ol><li>通过在20℃以14,000 xg离心离心样品20分钟洗脱聚糖。 </li><li>加入100μlPNG酶的在14,000rpm XG在20℃消化缓冲到过滤器，离心20分钟。收集含任何剩余的N洗-连接的聚糖，在相同的收集瓶作为洗脱剂。重复两次。在新的收集瓶删除过滤器和地点。 </li><li>孵育在-80℃的冰箱聚糖样品，直到冰冻（30-60分钟）。干燥完成后，在室温下，在真空中浓缩（4-6小时）。 注:N -glycans可以存储在-20℃用于衍生化之前长达六个月。 </li></ol> 4.蛋白质消化FASP 注意:如果蛋白质组学或脱酰胺化位点分析是不希望的，该协议的部分4可以被跳过。 <ol><li>冲洗ŧ他过滤，包含脱酰氨基肽，用400μl8M尿素缓冲。盖上收集瓶，轻轻漩涡混合。集中在过滤器上以14,000 xg离心15分钟。丢弃所有流量通过。 <ol><li>重复步骤4.1另外两次，总共三个尿素缓冲洗涤。 </li></ol></li><li>冲洗过滤器，将含有脱酰胺基的肽，用400μl的2M尿素，10毫氯化钙缓冲液（胰蛋白酶消化缓冲液）。盖上收集瓶，轻轻漩涡混合。集中在过滤器上以14,000 xg离心15分钟。丢弃所有流量通过。 <ol><li>重复步骤4.1两个附加次，一共有三个胰蛋白酶的消化缓冲洗涤。 </li><li>丢弃收集瓶洗一次完成。收集在一个新的收集瓶今后所有的洗脱液和洗涤。 </li></ol></li><li> （W / W）比率为发现或定量蛋白质组学的实验中，分别蛋白质:添加50微升猪改性胰蛋白酶在1:50或1:5胰蛋白酶。盖上收集瓶，轻轻漩涡混合。 </li><li>在37℃孵育样品2小时。 5胰蛋白酶:蛋白质比为1:50或1工作迅速，除去额外的50微升胰蛋白酶的样品和秒杀。轻轻涡样品1-2秒混合。孵育在50℃下将样品另外2小时。 </li><li>通过以14,000 xg离心纺丝15分钟洗脱的肽。 </li><li>从用400μl的1％的甲酸和0.001％两性洗涤剂3-16（骤冷缓冲液）的过滤器冲洗剩余的肽。通过在14000 XG纺纱15分钟洗脱过滤网。 </li><li>通过Bradford 19或BCA分析20量化样品中的肽浓度。 </li><li>孵育肽样品中的-80℃的冰箱冷冻前（30-60分钟）。干燥完成后，在室温下，在真空中浓缩（4-6小时）。 注:干肽可以储存在-20℃下长达三个月。 </li><li>重悬胰蛋白酶蛋白质只是之前的Liquið色谱-质谱法在2％乙腈（LC-MS）分析，98％的H 2 O和0.1％甲酸（流动相A），以所需的浓度。 2.5微升血浆胰蛋白酶范围肽浓度为100 - 300纳克/微升。 </li></ol> 5.衍生氮 -连接疏水酰肼标签聚糖<ol><li>重构干燥重（SIL）或光（NAT）P2GPN试剂在1ml 75:25的MeOH:乙酸（衍生溶液），为0.25毫克/毫升的最终浓度。试剂最多需要10分钟，充分溶解。广泛涡流以确保完全溶解。 注意:如果本衍生协议可与标准的N -glycans，与纯化的聚糖，P2GPN的摩尔比使用:聚糖应近似（以及不小于）17:1。 </li><li>标签干燥的N -glycans与SIL或NAT P2GPN试剂的200微升（50微克）。移液器上下重悬干聚糖。涡SAmples然后降速〜5秒的样品在台式离心机。 </li><li>与在56℃的试剂3小时反应的聚糖。 </li><li>立即从培养箱到真空浓缩移动聚糖。干以完成在55°C（3-5小时）。 注:此步骤淬火衍生反应;样品必须完全干燥，以防止交叉反应的后续步骤。 注意:干，标记样品可以储存在-20℃下长达六个月。 </li><li>悬浮只是LC-MS分析前标记ñ-glycans在25-50微升水中2 O的。移液器上下，以查看N -glycans完全溶解。 注:悬浮的体积依赖于糖蛋白的起始浓度，其可以从起始蛋白质浓度进行估计。然而，该范围将每个样品类型而有所不同，应单独优化。 </li><li>在14000 XG 5分钟离心样品。取出supernatant，小心不要让吸管尖触碰离心管的底部。 注意:过量的标签可能不可见的眼睛，而将通过离心被减少。 </li><li>结合的一对NAT和SIL样品，如果需要的话对于相对定量，以1:1的比例为串联分析。 </li></ol> 6.超高压液相色谱 - 质谱分析注:LC和MS为Easy NLC-1000和Q Exactive高场描述的条件，分别进行了优化，在内部进行蛋白质组学分析和聚糖分析21。这些条件可适于其他超高压敏或纳米液相色谱系统和其他高分辨力质谱仪但可能需要轻微的修改。利用高分辨率质谱法是必要的聚糖识别和分析脱酰胺22,23。 注意:步骤6.1-6.3能成为一个如果使用适当的反相色谱法或亲水相互作用商业陷阱和列kipped。 <ol><li>根据迈林等人的 24用作陷阱合成在100微米的ID毛细管的玻璃料。 </li><li>包在压力下的陷阱与2.6μM，100埃，C 18的包装材料，并切成5厘米的长度。 </li><li>收拾压力下的75微米的ID发射极柱2.6微米，100埃，C 18的包装材料，并切成30cm的长度。 </li><li>准备蛋白质组学（6.4.1）和糖组学（6.4.2）的工作流两个不同的LC-MS方法。 注:蛋白质和多糖样品可在任何顺序同样的陷阱/列设置运行。 <ol><li>对于蛋白质分析，注入〜400毫微克蛋白的上在流动相A（MPA）的列。运行在300标升/分钟的样品，根据表1，用1微升预柱平衡（500巴）和5微升分析柱平衡（500杆）。电离在表2中提供的MS条件下的蛋白质。 </li><li>对于聚糖分析，注入5微升的再悬浮，P2GPN标记的NAT / SIL等摩尔的样品上柱。运行在300标升/分钟的样品，根据表3，用2微升预柱平衡（500巴）和5微升分析柱平衡（500巴）。电离表4中提供的MS条件下聚糖。 </li></ol></li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 时间 </td><td> MPA％ </td><td> MPB％ </td></tr><tr><td> 0 </td><td> 100 </td><td> 0 </td></tr><tr><td>五</td><td> 98 </td><td> 2 </td></tr><tr><td> 105 </td><td> 80 </td><td> 20 </td></tr><tr><td> 135 </td><td> 68 </td><td> 32 </td></tr><tr><td> 136 </td><td>五</td><td> 95 </td></tr><tr><td> 151 </td><td>五</td><td> 95 </td></tr><tr><td> 152 </td><td> 100 </td><td> 0 </td></tr><tr><td> 167 </td><td> 100 </td><td> 0 </td></tr></tbody></table>进行表1中的LC条件蛋白质组学分析。梯度洗脱流动相B（MPB，98％乙腈，2％的H 2 O，0.1％甲酸）洗脱进行射击枪蛋白质组学，数据依赖性采集肽，MS实验。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="2"> MS 1参数 </td></tr><tr><td>质量范围（TH） </td><td> 375-1500 </td></tr><tr><td>解析度</td><td> 120000 </td></tr><tr><td> AGC </td><td> 1×10 6 </td></tr><tr><td>最大电离飞行时间质谱</td><td>三十</td></tr><tr><td> S-FR镜头水平</td><td> 55 </td></tr><tr><td>透射电镜毛细管P（℃） </td><td> 300 </td></tr><tr><td>喷雾电压</td><td> 1.75 </td></tr><tr><td colspan="2"> MS 2参数 </td></tr><tr><td>采集类型</td><td> TOP20 </td></tr><tr><td>解析度</td><td> 15000 </td></tr><tr><td> AGC </td><td> 1×10 5 </td></tr><tr><td>最大电离飞行时间质谱</td><td>三十</td></tr><tr><td>底部填充比</td><td> 2％ </td></tr><tr><td>隔离窗（TH） </td><td> 1.4 </td></tr><tr><td>充电状态排除</td><td> +1 </td></tr><tr><td>标准化碰撞能量</td><td> 27 </td></tr><tr><td>排除时间（s） </td><td> 20 </td></tr></tbody></table> 表2:使用高能量dissociatio MS条件蛋白质组学分析电喷雾电离的参数，MS 1采集和MS 2收购的轨道阱仪器。N（HCD）中给出。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 时间 </td><td> MPA％ </td><td> MPB％ </td></tr><tr><td> 0 </td><td> 95 </td><td>五</td></tr><tr><td> 1 </td><td> 70 </td><td>三十</td></tr><tr><td> 41 </td><td> 60 </td><td> 40 </td></tr><tr><td> 46 </td><td> 37 </td><td> 63 </td></tr><tr><td> 47 </td><td> 10 </td><td> 90 </td></tr><tr><td> 55 </td><td> 10 </td><td> 90 </td></tr><tr><td> 56 </td><td> 95 </td><td>五</td></tr><tr><td> 66 </td><td> 95 </td><td>五</td></tr></tbody></table> 表3:用于聚糖分析LC条件下进行梯度洗脱，以洗脱肼标记Ñ-glycans用于MS分析。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody> &lt;TR&gt; <td colspan="2"> MS 1参数 </td></tr><tr><td>质量范围（TH） </td><td> 600-1900 </td></tr><tr><td>解析度</td><td> 60000 </td></tr><tr><td> AGC </td><td> 5×10 5 </td></tr><tr><td>最大电离飞行时间质谱</td><td> 64 </td></tr><tr><td> S-FR镜头水平</td><td> 65 </td></tr><tr><td>毛细管温度（℃） </td><td> 325 </td></tr><tr><td>喷雾电压</td><td> 1.75 </td></tr><tr><td colspan="2"> MS 2参数 </td></tr><tr><td>采集类型</td><td> TOP12 </td></tr><tr><td>解析度</td><td> 15000 </td></tr><tr><td> AGC </td><td> 5×10 4 </td></tr><tr><td>最大电离飞行时间质谱</td><td> 100 </td></tr><tr><td>底部填充比</td><td> 1％ </td></tr><tr><td>隔离窗（TH） </td><td> 1.4 </td></tr><tr><td>修正了第一次大规模</tD&gt; <td> 125 </td></tr><tr><td>阶梯标准化碰撞能量</td><td> 10/20/30 </td></tr><tr><td>排除时间（秒） </td><td> 15 </td></tr></tbody></table> 表4:MS条件酰肼标记ñ-glycan分析电喷雾电离的参数，MS 1采集和MS 2收购使用高能量解离（HCD）的轨道阱仪器给出。 7.蛋白质组学和脱酰胺分析<ol><li>确定使用标准的生物信息学搜索工具的蛋白/多肽。 注意:下面的分析协议用在UniprotKB的SWISS-PROT / TrEMBL的数据库，并在蛋白质组发现者1.4软件通过SEQUEST搜索设计，然而，该协议可以直接转换为使用GUI软件的任何蛋白质搜索和评分引擎。下面给出的所有命令可以软件接口通过下拉菜单来访问。 <ol> &lt;li&gt;创建或由Apweiler 等人从基因组数据或提供蛋白质组数据库下载一个有机体适当蛋白质序列数据库。25 注:通用蛋白质组数据库包括SWISS-PROT，TrEMBL的，和NCBI，而是可以从内部数据构建为好。 </li><li>内的软件中，选择一个数据库的搜索引擎，并选择参数，根据所获得的数据的质量和所需的搜索的严谨，如Perkins 等 人所述。对于MASCOT 26或主机等为SEQUEST 27。 <ol><li>对于高精确质量数据，在软件搜索设置参数如下:最大2错过了分裂，5ppm的前驱物质量偏差，以及0.02大片段质量偏差（优化的精确脱酰胺检测22），并包括以下胰肽修改:"carbamidomethyl（C）的"静态修改和"脱酰胺（N / Q）"和"牛idation（M）"动态修改。如果使用18 O消化标签，包括"脱酰胺18 O（1）"作为一个附加动态修改。 </li><li>搜索，使用所选择的发动机，对蛋白质数据库（7.1.1）的原始肽数据。 注:质量公差应适合所用仪器，而不是任意低。 注意:搜索功能通过使用各种搜索引擎的专用算法软件自动完成。该过滤器算法（MASCOT），SEQUEST或算法其他组合被用来从肽鉴定蛋白质和得分命中的有效性;上可用的工具的更多信息覆盖从冈萨雷斯Galarza 等 28,29审查。根据软件，附加的搜索参数，可能需要根据用户的判断来选择。 </li></ol></li><li>导出确定肽手动策展和菲尔特呃分析。 </li></ol></li><li>手动策划包含在N的天冬酰胺所识别的脱酰胺的肽的列表-连接的糖基化基序（NX-（S / T），其中X≠P）。交叉验证这些网站与文献已知的糖基化的图案和创造业绩的两个池（验证和理论）。 </li><li>使用光谱计30通过比较脱酰胺和非脱酰胺基形式的丰度到给定的糖基化位点的百分比占用比较。 </li></ol> 8.聚糖相对定量注:以下定性和定量是使用的Xcalibur软件完成。然而，用于分析原始数据并自动任何软件集成色谱峰可以被取代。 <ol><li>产生从己糖，脱氧己糖，氨基己糖，唾液酸的生物可能组合聚糖组合物的理论列表，其它山ccharides。为了这个目的，一个人的聚糖数据库已由Walker等 15日公开，并且可以原样使用。对于理论上的数据库，物种特异性的生物学约束必须在组合空间的罚款 ​​，而这些规则是由Kronewitter 等 31描述。 </li><li>计算从原子量聚糖单同位素质量（M）为质子电荷态+ 1-3各组成。修改单一同位素群众包括增加的NAT（254.14191克/摩尔）或SIL（260.162042克/摩尔）P2GPN标签。 </li><li>打开在的Xcalibur路线图视图中的"处理设置"程序。使用在含有理论单同位素质量为步骤8.2中产生的列表中设置为正好在补充材料从Walker 等人描述的处理方法。15 [M + H] 1+，[M + 2H] 2+和[ M + 3H] 3+的物种。 注意:要确认正确超越身份聚糖质量，支持NAT / SIL双工运行，检查NAT和SILñ-glycan对共流出具有相同的保留时间。定性，交验证标识与MS 2谱，必须包含属于氧鎓离子系列32的峰。 </li><li>导出集成，提取离子色谱（XIC）区域到Excel得到为正好在补充材料从Walker 等人所述的SIL和NAT丰度。15 </li><li>在Excel中，程序中的新列的单元格，以通过调整SIL丰度根据由Walker 等人发表的等式计算用于分子量重叠的修正1 5: <img alt="式（1）" src="/files/ftp_upload/53735/53735eq1.jpg" /> ， 其中，A是单同位素峰与理论同位素重叠， <img alt="公式（2）" src="/files/ftp_upload/53735/53735eq2.jpg" />可能每个成分单独计算。 </li><li>在第二列中，程序中的细胞使用公式总归聚糖因子（TGNF），每光谱正常化的NAT和SIL渠道，如Walker 等人所述1 5: <img alt="公式3" src="/files/ftp_upload/53735/53735eq3.jpg" /> ， 其中N是数N -glycans上述定量限。 </li><li>在新的一列，程序中的细胞取SIL的比率:NAT聚糖（或反之亦然）来计算在两个样本之间的浓度倍数变化。 </li></ol>

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We have a collaborative relationship with Cambridge Isotope Laboratories (CIL) to disseminate the P2GPN reagent and technology to the broader scientific community in the form of a product (GTK-1000). However, we do not receive any revenue based on the sale of this product.

High-throughput quantitative methods are needed to facilitate routine glycan analysis. For the last thirty years, glycomics analysis has been limited to a subset of research groups, despite its importance in disease, clinical applications, and pharmaceuticals. The FANGS-P2GPN purification and tagging method for glycomics and proteomics performs the same analysis on a single aliquot of sample, reducing the cost of supplies and the amount of material needed (particularly important in human and mouse studies). Furthermore, efforts to minimize variability in preparations are critically important, as every additional step contributes to error, potentially masking important but low-abundant changes in case-control studies. Coupling of FANGS to hydrophobic hydrazide tagging allows protein and glycan samples to be run on the same RPLC column, enhances glycan ionization, provides for relative quantification, and can be quantitatively applied to plasma.
For N-glycan analysis, it is critical to use the suggested level of PNGase F to achieve full de-glycosylation. Though glycans are solvent exposed, denaturation of proteins and excess enzyme help ensure efficient and complete cleavage. For accurate quantitation of the glycans, it is necessary to ensure that they are completely dried after derivatization to quench the reaction and prevent cross-reactions when mixing the NAT and SIL species. Finally, when extending the workflow to glycosite analysis, timing of the steps is critical to minimize non-specific deamidation. The modified protocols provided for combined glycomics and proteomics analysis work consistently when performed accordingly.
The workflow achieves accurate relative quantitation of N-glycans from plasma compared to the gold-standard, SPE method. There is no apparent bias in the types of glycans extracted in terms of molecular weight, hydrophilicity, and compositional structure. Though we have not explored the qualitative analysis of O-linked glycans, we expect that FANGS could accommodate the addition of a &#946;-elimination step post-PNGase F digestion of N-linked glycans. However, procedures would require significant modification for reagent cleanup prior to mass spectrometry, and peptide analysis will be significantly impacted. For proteomics, the same depth of proteome coverage is achieved compared to traditional FASP methods. Importantly, methods achieve a minimal false discovery rate for N-glycan deamidation. While the method is compatible with 18O labeling of Asn during the PNGase digestion step22,23, the low glycosylation site false discovery rate suggests that it may not be necessary, further reducing costs and complexity.
The proteome is not enriched for glycoproteins in this method, which has both advantages and disadvantages. Certain low abundant glycoproteins may not be detected in the analysis. However, the occupancy of glycosylated sites per protein, can be compared between biological samples. Additionally, the error and bias introduced from lectin affinity purification or chemical enrichment is eliminated. In conclusion, coupling of FANGS to the individuality normalization when labeling with glycan hydrazide tags strategy results in a simplified, quantitative, high-throughput method for the tandem analysis of the glycome and proteome with great potential for application in clinical case-control studies.

在蛋白质组学领域，滤波器计算机辅助样品制备（FASP）已被广泛地用于其以最小化起始材料的量，降低样品制备的工件，并最大限度地提高样品通量1能力通过。然而，这样的方法还没有出现，并获得牵引力为糖组学的领域。高通量的发展，正在由于糖基化的生物防御的积分作用和由癌症或疾病2,3-其调制所需要定量的工作流程。在哺乳动物中，N- ​​-glycans是由重复糖单元（己糖（十六进制），氨基己糖（HexNac），唾液酸（的NeuAc），和岩藻糖（Fuc）），其布置的芯结构（六角3 HexNac 2）4共价结合的以天冬酰胺。虽然当异构体的计数glycospace是相当大的（&gt; 10 12），它是相当小的一个组合物的基础上和分子量典型地为1,000-8,000达5 &lt;范围/ SUP&gt;。类的组成均一性和聚糖的亲水性提出了独特的挑战，以净化，分 ​​离，和质谱（MS）的工作流程6。 传统上，N- -glycans从蛋白质或肽通过肽Ñ消化糖苷酶F（PNG酶F），然后通过凝集素亲和层析7富集，由酰肼珠8捕获，或通过固相萃取（SPE）9,10纯化。虽然这些方法都是高度有效的，它们引入脱盐额外的步骤，限制同时处理的样本的数目。在过去的十年中，许多用于糖组学的高通量平台已经被提出。 Kim等人发表使用真空操作时，SPE 96孔板11的半自动化方法。或者，亲和过滤方法（N- -glyco-FASP）由曼基，其所需的网络连接的初始衍生开发滤波器与凝集素12的复合物。最后，托马斯-奥茨组提出了一个半定量的方法，辅助ñ-Glycan分离（毒牙）过滤器，它利用了glycospace 13的窄组成的大小。依靠分子量截止的过滤器，小的污染物被第一洗涤浪费，然后N个 -glycans消化和洗脱。去糖基化蛋白保留在过滤器上在本协议中，可以进行在线FASP。 鉴定并通过电喷雾电离聚糖的定量（ESI）MS需要离线分离的（部分）号决议，同分异构体和衍生的检测低丰度的物种。标记在个性与聚糖肼标记战略正火时赋予与反相液相层析（RPLC）14,15的兼容性。 4-苯乙基 - 苯甲酰肼（P2PGN）疏水标签介导的聚糖的亲水性，ENH由平均ancing电离，四倍16。在轻便条件1化学计量:虽然其他技术，如全甲基17或胺反应性标记化学18，提供了类似的优点，在酰肼反应，聚糖反应1。相对定量通过用天然（NAT）或13 的 C 6稳定的同位素标签（SIL）的衍生样品的串联分析来实现的。 下面的方法演变等离子应用和夫妇它的精确的相对定量P2GPN疏水标记的獠牙。此外，它的目的是在样品的单一等份执行射击枪蛋白质组学，脱酰胺剖析和定量糖组学，而不损害分析的完整性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Acetic Acid (50%):&#160;</td>
			<td >Sigma Aldrich&#160;</td>
			<td >45754</td>
			<td ></td>
		</tr>
		<tr >
			<td >Acetonitrile, HPLC grade</td>
			<td >Burdick &#38; Jackson&#160;</td>
			<td >AH015-4</td>
			<td ></td>
		</tr>
		<tr >
			<td >Ammonium Bicarbonate</td>
			<td >Sigma Aldrich&#160;</td>
			<td >A6141</td>
			<td ></td>
		</tr>
		<tr >
			<td >Bradford Reagent</td>
			<td >Sigma Aldrich&#160;</td>
			<td >B6916</td>
			<td>Alternative: Bicinchoninic acid kit (Sigma Aldrich BCA1)</td>
		</tr>
		<tr >
			<td >Calcium chloride</td>
			<td >Sigma Aldrich&#160;</td>
			<td >C1016</td>
			<td></td>
		</tr>
		<tr >
			<td >Centrifuge</td>
			<td >Eppendorf</td>
			<td >5804 R</td>
			<td >Alternate centrifuges that reach 14,000 x g are suitable</td>
		</tr>
		<tr >
			<td >DL-Dithiothreitol, 1M in solution</td>
			<td >Sigma Aldrich&#160;</td>
			<td >646563</td>
			<td ></td>
		</tr>
		<tr >
			<td >Easy-nLC 1000</td>
			<td >Thermo Scientific</td>
			<td >LC120</td>
			<td >Alternate nano or ultra high pressure LCs will produce similar data, such as: 1. Dionex UltiMate&#210; 3000 LC (Thermo Scientific) 2. Acquity UPLC (Waters)</td>
		</tr>
		<tr >
			<td >Floating Tube Rack</td>
			<td >TedPella</td>
			<td >20831-20</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetuin</td>
			<td >New England Biolabs&#160;</td>
			<td >P6042S</td>
			<td ></td>
		</tr>
		<tr >
			<td >Fisher Scientific Isotemp Standard Lab Ovens</td>
			<td >Fisher Scientific</td>
			<td >11-690-625F</td>
			<td >Alternate incubators that reach 56 &#176;C are suitable</td>
		</tr>
		<tr >
			<td >Formic Acid</td>
			<td >Sigma Aldrich&#160;</td>
			<td >56302</td>
			<td ></td>
		</tr>
		<tr >
			<td >GE Microwave Oven</td>
			<td >General Electric</td>
			<td >57B5 E82904</td>
			<td >Any microwave with adjustable power settings is suitable</td>
		</tr>
		<tr >
			<td >INLIGHT Glycan Tagging Kit&#160;</td>
			<td >Cambridge Isotope Laboratories</td>
			<td >GTK-1000</td>
			<td >The INLIGHT kit provides NAT and SIL versions of the P2GPN reagent.</td>
		</tr>
		<tr >
			<td >Iodoacetamide</td>
			<td >Sigma Aldrich&#160;</td>
			<td >A3221</td>
			<td></td>
		</tr>
		<tr >
			<td >Kinetix 2.6 mM, 100 &#197;, C18 bulk stationary phase</td>
			<td >Phenomenex&#160;</td>
			<td>Bulk Media</td>
			<td >Alternative: Any C18 stationary phase &#163; 5 mM&#160;</td>
		</tr>
		<tr >
			<td >Mascot Daemon Software and Server</td>
			<td >Matrix Science</td>
			<td ></td>
			<td >Alternative: Proteome Discoverer Software (Thermo Scientific)</td>
		</tr>
		<tr >
			<td >Methanol, HPLC grade</td>
			<td >Burdick &#38; Jackson&#160;</td>
			<td >AH230-4</td>
			<td ></td>
		</tr>
		<tr >
			<td >PicoFrit Self-Pack Column: 360 um, OD 75um ID, 15 um tip, non-coated, 5 per box, 50 cm</td>
			<td >New Objective</td>
			<td >1 5 PF360-75-15-N-5</td>
			<td></td>
		</tr>
		<tr >
			<td >PNGase F (glycerol-free), 75,000 units/ml</td>
			<td >New England BioLabs&#160;</td>
			<td >P0705L</td>
			<td ></td>
		</tr>
		<tr >
			<td >Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer</td>
			<td >Thermo Scientific</td>
			<td ></td>
			<td >Alternate high mass accuracy (&#163; 5 ppm) mass spectrometers will provide similar data</td>
		</tr>
		<tr >
			<td >RNase B</td>
			<td >New England Biolabs&#160;</td>
			<td >P7817S</td>
			<td ></td>
		</tr>
		<tr >
			<td >Trypsin from Porcine Pancreas</td>
			<td >Sigma Aldrich&#160;</td>
			<td >T6567-5X</td>
			<td></td>
		</tr>
		<tr >
			<td >Urea</td>
			<td >Sigma Aldrich&#160;</td>
			<td >51456</td>
			<td></td>
		</tr>
		<tr >
			<td >Vacuum ConcentratorSavant SPD131DDA SpeedVac Concentrator</td>
			<td >Thermo Scientific</td>
			<td >SPD131DDA</td>
			<td >Alternate vacuum concentrators are suitable</td>
		</tr>
		<tr >
			<td >Vivacon 500 30 kDa Filters&#160;</td>
			<td >Sartorius Stedim Biotech</td>
			<td >VN01H22</td>
			<td >Alternative: Amicon Ultra 0.5 Centrifugal Filter Units with Ultracel-10 kDa Membrane (Millipore UFC501096)</td>
		</tr>
		<tr >
			<td >Water, HPLC grade</td>
			<td >Burdick &#38; Jackson&#160;</td>
			<td >AH365-4</td>
			<td ></td>
		</tr>
		<tr >
			<td >Water, 18O</td>
			<td >Cambridge Isotope Laboratories</td>
			<td >OLM-240-97-1</td>
			<td >The addition of 18O in the PNGase F digest step is optional and may not be necessary for deamidation studies completed with 95% confidence</td>
		</tr>
		<tr >
			<td >Xcalibur 2.0</td>
			<td >Thermo Scientific</td>
			<td>XCALIBUR20</td>
			<td></td>
		</tr>
		<tr >
			<td >Zwittergent Test Kit</td>
			<td >Merck Millipore</td>
			<td >693030</td>
			<td></td>
		</tr>
	</tbody>
</table>

a quantitative glycomics and proteomics combined purification strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

<img alt="图1" src="/files/ftp_upload/53735/53735fig1.jpg" /> 图1:毒牙，P2GPN疏水标记耦合方法的结合蛋白质组学和糖组学分析方案（方法A）给出只有糖组学处理和串联糖组学和蛋白质组学分析，糖基化位点的识别是不同的步骤，突出显示。 <a href="https://www.jove.com/files/ftp_upload/53735/53735fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 一字形蛋白质组学和糖组学，基于过滤器的P2GPN疏水标签方法（方法A）使用合并的母鸡血浆样品（ 图1）进行了验证识别，定量，和分子量偏压。全权糖组学试验，以N的丰度相互比较-glycans通过方法萃取A到carbograph SPE（金标准，方法B）作了（ 图2）。有在这两种方法之间的聚糖的丰度没有显著差异。用同样的方法提取的聚糖的NAT的比率:帧内变异也通过量化SIL评估。这两种方法的日志2分布为高斯，集中于零，而不是显著不同（ 图3A-B）。分子量范围的两个协议完全重叠之间，这表明使n -glycans根据分子量范围和亲水性（ 图4）的过滤器也没有区别。 <img alt="图2" src="/files/ftp_upload/53735/53735fig2.jpg" /> 图2:NAT和SIL n的等摩尔混合物-glycans通过方法A或方法B中提取从母鸡血浆2.5微升（N = 4）将样品肛门制备根据第6条每个聚糖的丰度提出建议的参数，每个NAT / SIL通道由UPLC-MS yzed，被提取的离子色谱图（MMA = 3 PPM）整合下的面积计算，校正分子重重叠，以及调整由TGNF。这些比率示与它们的标准误差。方法B的丰度:方法A N -glycans没有显著差异（P&gt; 0.05）。 <a href="https://www.jove.com/files/ftp_upload/53735/53735fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53735/53735fig3.jpg" /> 图3:内部变异性（A）方法A（N = 133）或（B）方法B（N = 123）的策略进行比较。 NAT和SILÑ-glycans的等摩尔混合物，从同样的提取方案，在三个技术重复进行了分析。日志2分布集中于零，并没有显著不同的两个工作流程之间。 <a href="https://www.jove.com/files/ftp_upload/53735/53735fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53735/53735fig4.jpg" /> 图4:从每个策略聚糖的分子量范围进行比较。这两个工作流程，取得了跨越聚糖相同的分子量范围内。在一个协议-glycans检测N对其他的下跌只是上面的检测（1×10 5丰度）的限制，并不反映系统偏差。 <a href="https://www.jove.com/files/ftp_upload/53735/53735fig4large.jpg" target="_blank">请点击此处查看大图版本这个数字。</a> 表5）传统的FASP制剂。我们的典型18小时滤波器基于酶F消化，随后FASP胰蛋白酶消化（标准协议）导致非特异性脱酰胺显著水平，这可与glycosites的识别干扰。因此，利用在升高的温度（50℃）和一个酶F尖峰在步骤（SPI协议）一个较短的温育时间的方法进行了探讨作为替代，用微波消解协议（MD协议）沿，以最小化非具体的脱酰胺。在新的消解方法观察到的聚糖不是从标准的18小时组成或丰度显著不同，37℃过滤酶F消化（ <strong&gt;图5）。具有短胰蛋白酶温育相结合，非特异性脱酰胺是显著减少，与glycosites &lt;5％（ 表5）的假阳性率。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="2"></td><td colspan="4">蛋白质</td><td></td><td colspan="4">肽</td><td></td><td colspan="4">脱酰胺肽</td><td></td><td colspan="4"> Glycosites </td><td></td><td colspan="4">糖蛋白</td></tr><tr><td colspan="2"></td><td colspan="2"> + </td><td colspan="2"> - </td><td></td><td colspan="2"> + </td><td colspan="2"> - </td><td></td><td colspan="2"> + </td><td colspan="2"> - </td><td></td><td colspan="2"> + </td><td colspan="2"> - </td><td></td><td colspan="2"> + </td><td colspan="2"> - </td></tr><tr><td colspan="2"> FASP </td><td colspan="2"></td><td colspan="2"&gt; 249 </td><td></td><td colspan="2"></td><td colspan="2"> 3083 </td><td></td><td colspan="2"></td><td colspan="2"> 795 </td><td></td><td colspan="2"></td><td colspan="2">五</td><td></td><td colspan="2"></td><td colspan="2">五</td></tr><tr><td colspan="2"> 18小时</td><td colspan="2"> 217 </td><td colspan="2"> 304 </td><td></td><td colspan="2"> 6013 </td><td colspan="2"> 5086 </td><td></td><td colspan="2"> 1029 </td><td colspan="2"> 733 </td><td></td><td colspan="2"> 257 </td><td colspan="2"> 24 </td><td></td><td colspan="2"> 112 </td><td colspan="2"> 18 </td></tr><tr><td colspan="2"> MD </td><td colspan="2"> 270 </td><td colspan="2"> 266 </td><td></td><td colspan="2"> 5190 </td><td colspan="2"> 5125 </td><td></td><td colspan="2"> 465 </td><td colspan="2"> 455 </td><td></td><td colspan="2"> 254 </td><td colspan="2"> 8 </td><td></td><td colspan="2"> 102 </td><td colspan="2"> 7 </td></tr><tr><td colspan="2"> SPI </td><td colspan="2"> 281 </td><td colspan="2"> 288 </td><td></td><td colspan="2"> 4729 </td><td colspan="2"> 4482 </td><td></td><td colspan="2"> 602 </td><td colspan="2"> 573 </td><td></td><td colspan="2"> 232 </td><td colspan="2"> 10 </td><td></td><td colspan="2"> 145 </td><td colspan="2"> 8 </td></tr></tbody></table> 表 5: 从标准的胰蛋白酶蛋白质组数据的比较消化相对于直列尖牙-P2GPN标记方法进行了准备，用进行（+）和无（ - ）PNG酶F到测定非特异性脱酰胺和估计的背景率glycosite误报率。蛋白质鉴定用1％的假发现率（FDR）;根据蛋白质组发现者1.4"高"肽信心过滤肽（Q &lt;0.01）; glycosites被identifi基于包含保守的糖基化基序（NXS / T）的脱酰胺作用的独特序列编;和糖蛋白被定义为含有至少一个被识别glycosite鉴定的蛋白质。 <img alt="图5" src="/files/ftp_upload/53735/53735fig5.jpg" /> 图5:缩短为聚糖协议被开发，以减少样品中的脱氨和相比，18小时的尖牙酶F消化。在丰度的平均差异分别为10％和5％的MD（2.2.3）和SPI（2.2.2）协议，分别。确定了48聚糖，90％显示小于1.5倍的差异。 <a href="https://www.jove.com/files/ftp_upload/53735/53735fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Quantitative Glycomics and Proteomics Combined Purification Strategy at JoVE.com

A Quantitative Glycomics and Proteomics Combined Purification Strategy

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

定量糖组学和蛋白质组学组合式净化策略

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

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14.8K 次观看.  North Carolina State University. A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

视频: A Quantitative Glycomics and Proteomics Combined Purification Strategy

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

规模以上非靶向代谢组学剖析血清超高效液相色谱 - 质谱（UPLC-MS）

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

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化学

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

定量蛋白质组学使用还原二甲基化的稳定同位素标记

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

Synthesis and Purification of Iodoaziridines Involving Quantitative Selection of the Optimal Stationary Phase for Chromatography

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. Diiodomethyllithium is prepared by the deprotonation of diiodomethane with LiHMDS, in a THF/diethyl ether mixture, at -78 &#176;C in the dark. These conditions are essential for the stability of the LiCHI2 reagent generated. The subsequent dropwise addition of N-Ts aldimines to the preformed diiodomethyllithium solution affords an amino-diiodide intermediate, which is not isolated. Rapid warming of the reaction mixture to 0 &#176;C promotes cyclization to afford iodoaziridines with exclusive cis-diastereoselectivity. The addition and cyclization stages of the reaction are mediated in one reaction flask by careful temperature control.
Due to the sensitivity of the iodoaziridines to purification, assessment of suitable methods of purification is required. A protocol to assess the stability of sensitive compounds to stationary phases for column chromatography is described. This method is suitable to apply to new iodoaziridines, or other potentially sensitive novel compounds. Consequently this method may find application in range of synthetic projects. The procedure involves firstly the assessment of the reaction yield, prior to purification, by 1H NMR spectroscopy with comparison to an internal standard. Portions of impure product mixture are then exposed to slurries of various stationary phases appropriate for chromatography, in a solvent system suitable as the eluent in flash chromatography. After stirring for 30 min to mimic chromatography, followed by filtering, the samples are analyzed by 1H NMR spectroscopy. Calculated yields for each stationary phase are then compared to that initially obtained from the crude reaction mixture. The results obtained provide a quantitative assessment of the stability of the compound to the different stationary phases; hence the optimal can be selected. The choice of basic alumina, modified to activity IV, as a suitable stationary phase has allowed isolation of certain iodoaziridines in excellent yield and purity.

A protocol for the diastereoselective one-pot preparation of cis-N-Ts-iodoaziridines is described. The generation of diiodomethyllithium, addition to N-Ts aldimines and cyclization of the amino gem-diiodide intermediate to iodoaziridines is demonstrated. Also included is a protocol to rapidly and quantitatively assess the most appropriate stationary phase for purification by chromatography.

合成和Iodoaziridines纯化涉及定量选择最优固定相的色谱

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. ...

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

改善凝胶还原β-消除综合O连接和磺基糖组学质谱

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

CN-GELFrEE  - 透明天然凝胶洗脱液体馏分偷天陷阱电泳

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding ...

Glycan Node Analysis: A Bottom-up Approach to Glycomics

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and extracellular events. Many pathological conditions, notably cancer, affect gene expression, which can in turn deregulate the relative abundance and activity levels of glycoside hydrolase and glycosyltransferase enzymes. Unique aberrant whole glycans resulting from deregulated glycosyltransferase(s) are often present in trace quantities within complex biofluids, making their detection difficult and sometimes stochastic. However, with proper sample preparation, one of the oldest forms of mass spectrometry (gas chromatography-mass spectrometry, GC-MS) can routinely detect the collection of branch-point and linkage-specific monosaccharides ("glycan nodes") present in complex biofluids. Complementary to traditional top-down glycomics techniques, the approach discussed herein involves the collection and condensation of each constituent glycan node in a sample into a single independent analytical signal, which provides detailed structural and quantitative information about changes to the glycome as a whole and reveals potentially deregulated glycosyltransferases. Improvements to the permethylation and subsequent liquid/liquid extraction stages provided herein enhance reproducibility and overall yield by facilitating minimal exposure of permethylated glycans to alkaline aqueous conditions. Modifications to the acetylation stage further increase the extent of reaction and overall yield. Despite their reproducibility, the overall yields of N-acetylhexosamine (HexNAc) partially permethylated alditol acetates (PMAAs) are shown to be inherently lower than their expected theoretical value relative to hexose PMAAs. Calculating the ratio of the area under the extracted ion chromatogram (XIC) for each individual hexose PMAA (or HexNAc PMAA) to the sum of such XIC areas for all hexoses (or HexNAcs) provides a new normalization method that facilitates relative quantification of individual glycan nodes in a sample. Although presently constrained in terms of its absolute limits of detection, this method expedites the analysis of clinical biofluids and shows considerable promise as a complementary approach to traditional top-down glycomics.

This article presents an enhanced form of a novel bottom-up glycomics technique designed to analyze the pooled compositional profile of glycans in unfractionated biofluids through the chemical breakdown of glycans into their constituent linkage-specific monosaccharides for detection by GC-MS. Potential applications include early detection of cancer and other glycan-affective disorders.

聚糖节点分析:自下而上的方法，以糖原组学

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and ...

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

A protocol for the protein quantification in complex biological fluids using automated immuno-MALDI (iMALDI) technology is presented.

多肽和蛋白质量化使用自动免疫-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a ...

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good&#39;s buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera.

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

In this work, a protocol for signal enhancement of nanoprobe-based biosensing is presented. The protocol is based on the reduction of chloroauric acid onto the surface of existing nanoprobes that consist of gold, silver, silica or iron-oxide nanoparticles.

原位金纳米粒子合成快速 Nanoprobe 信号增强

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity ...

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

利用毛细管区电泳串联质谱技术进行大尺度自上而下蛋白质组学

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down ...

Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers

Routine production of radiotracers used in positron emission tomography (PET) mostly relies on wet chemistry where the radioactive synthon reacts with a non-radioactive precursor in solution. This approach necessitates purification of the tracer by high performance liquid chromatography (HPLC) followed by reformulation in a biocompatible solvent for human administration. We recently developed a novel 11C-methylation approach for the highly efficient synthesis of carbon-11 labeled PET radiopharmaceuticals, taking advantage of solid phase cartridges as disposable &#34;3-in-1&#34; units for the synthesis, purification and reformulation of the tracers. This approach obviates the use of preparative HPLC and reduces the losses of the tracer in transfer lines and due to radioactive decay. Furthermore, the cartridge-based technique improves synthesis reliability, simplifies the automation process and facilitates compliance with the Good Manufacturing Practice (GMP) requirements. Here, we demonstrate this technique on the example of production of a PET tracer Pittsburgh compound B ([11C]PiB), a gold standard in vivo imaging agent for amyloid plaques in the human brains.

Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers

We report an efficient carbon-11 radiolabeling technique to produce clinically relevant tracers for Positron Emission Tomography (PET) using solid phase extraction cartridges. 11C-methylating agent is passed through a cartridge preloaded with precursor and successive elution with aqueous ethanol provides chemically and radiochemically pure PET tracers in high radiochemical yields.

用于生产PET示踪剂的固体阶段11C甲基化、纯化和配方

Routine production of radiotracers used in positron emission tomography (PET) mostly relies on wet chemistry where the radioactive synthon reacts with a ...