Name: a quantitative glycomics and proteomics combined purification strategy
Uploaded: 2016-03-08T14:00:00.000+00:00
Duration: 11 min 38 s
Description: Watch this Scientific Journal Video about A Quantitative Glycomics and Proteomics Combined Purification Strategy at JoVE.com

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><tbody><tr><td>Acetic Acid (50%)</td><td>Sigma Aldrich&amp;nbsp;</td><td>45754</td><td></td></tr><tr><td>Acetonitrile, HPLC grade</td><td>Burdick &amp;amp; Jackson&amp;nbsp;</td><td>AH015-4</td><td></td></tr><tr><td>Ammonium Bicarbonate</td><td>Sigma Aldrich&amp;nbsp;</td><td>A6141</td><td></td></tr><tr><td>Bradford Reagent</td><td>Sigma Aldrich&amp;nbsp;</td><td>B6916</td><td>Alternative: Bicinchoninic acid kit (Sigma Aldrich BCA1)</td></tr><tr><td>Calcium chloride</td><td>Sigma Aldrich&amp;nbsp;</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, 1&amp;nbsp;M in solution</td><td>Sigma Aldrich&amp;nbsp;</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&amp;Ograve; 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&amp;nbsp;</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&amp;nbsp;&amp;deg;C are suitable</td></tr><tr><td>Formic Acid</td><td>Sigma Aldrich&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>A3221</td><td></td></tr><tr><td>Kinetix 2.6 mM, 100 &amp;Aring;, C&lt;sub&gt;18&lt;/sub&gt; bulk stationary phase</td><td>Phenomenex&amp;nbsp;</td><td>Bulk Media</td><td>Alternative: Any C&lt;sub&gt;18&lt;/sub&gt; stationary phase 5&amp;nbsp;mM</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 &amp;amp; Jackson&amp;nbsp;</td><td>AH230-4</td><td></td></tr><tr><td>PicoFrit Self-Pack Column: 360&amp;nbsp;&amp;#956;m, OD 75&amp;nbsp;&amp;#956;m ID, 15&amp;nbsp;&amp;#956;m 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&amp;nbsp;units/ml</td><td>New England BioLabs</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 (5&amp;nbsp;ppm) mass spectrometers will provide similar data</td></tr><tr><td>RNase B</td><td>New England Biolabs&amp;nbsp;</td><td>P7817S</td><td></td></tr><tr><td>Trypsin from Porcine Pancreas</td><td>Sigma Aldrich&amp;nbsp;</td><td>T6567-5X</td><td></td></tr><tr><td>Urea</td><td>Sigma Aldrich&amp;nbsp;</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&amp;nbsp;</td><td>Sartorius Stedim Biotech</td><td>VN01H22</td><td>Alternative: Amicon Ultra 0.5 Centrifugal Filter Units with Ultracel-10&amp;nbsp;kDa Membrane (Millipore UFC501096)</td></tr><tr><td>Water, HPLC grade</td><td>Burdick &amp;amp; Jackson&amp;nbsp;</td><td>AH365-4</td><td></td></tr><tr><td>Water, &lt;sup&gt;18&lt;/sup&gt;O</td><td>Cambridge Isotope Laboratories</td><td>OLM-240-97-1</td><td>The addition of &lt;sup&gt;18&lt;/sup&gt;O 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 ...

a-quantitative-glycomics-and-proteomics-combined-purification-strategy

Research

JoVE Journal

Chemistry

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

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 ...

科研

化学

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

用等压标记、广泛的液相色谱法、质谱法和软件辅助定量技术进行深层蛋白质组分析

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

生物化学

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

通过肽浓缩和质谱检测蛋白质泛化位点检测

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

生物学

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and determine glycan alterations in diseases. Most commercially available biopharmaceutical proteins are glycoproteins. The efficacy of these drugs is affected by glycosylation patterns. Therefore, an in-depth characterization method for the N-glycans is necessary. Here, we present a comprehensive approach for qualitative and quantitative analysis of N-glycans using hydrophilic interaction liquid chromatography equipped with fluorescence detection and tandem mass spectrometry (HILIC-FLD-MS/MS). N-glycans were released from glycoproteins with a facile method and labeled by a procainamide fluorophore tag in the strategy. Subsequently, the procainamide labeled N-glycans were analyzed by a HILIC-FLD-MS/MS technique. In this approach, N-glycan structures were confirmed by the tandem mass spectrometric analysis, whereas fluorescence detection was used for the quantitative analysis. An application for data analysis of the detected N-glycan peaks is described in the study. This protocol can be applied to any glycoprotein extracted from various species.

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

N-glycan profiling of glycoproteins is essential for discovering novel biomarkers and understanding glycan functions in cellular events. Additionally, N-glycan analysis of protein biopharmaceuticals is very important for human use. In this current article, a high-throughput strategy for identifying and quantifying N-glycan structures was presented using the HILIC-FLD-MS/MS technique.

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and ...

Organic Solvent-Based Protein Precipitation for Robust Proteome Purification Ahead of Mass Spectrometry

While multiple advances in mass spectrometry (MS) instruments have improved qualitative and quantitative proteome analysis, more reliable front-end approaches to isolate, enrich, and process proteins ahead of MS are critical for successful proteome characterization. Low, inconsistent protein recovery and residual impurities such as surfactants are detrimental to MS analysis. Protein precipitation is often considered unreliable, time-consuming, and technically challenging to perform compared to other sample preparation strategies. These concerns are overcome by employing optimal protein precipitation protocols. For acetone precipitation, the combination of specific salts, temperature control, solvent composition, and precipitation time is critical, while the efficiency of chloroform/methanol/water precipitation depends on proper pipetting and vial manipulation. Alternatively, these precipitation protocols are streamlined and semi-automated within a disposable spin cartridge. The expected outcomes of solvent-based protein precipitation in the conventional format and using a disposable, two-stage filtration and extraction cartridge are illustrated in this work. This includes the detailed characterization of proteomic mixtures by bottom-up LC-MS/MS analysis. The superior performance of SDS-based workflows is also demonstrated relative to non-contaminated protein.

The present protocol describes solvent-based protein precipitation under controlled conditions for robust and rapid recovery and purification of proteome samples prior to mass spectrometry.

有机溶剂基蛋白质沉淀，在质谱分析之前实现强大的蛋白质组纯化

While multiple advances in mass spectrometry (MS) instruments have improved qualitative and quantitative proteome analysis, more reliable front-end ...

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the specific roles of glycans, the relationship between their structures and their impact on the functions of proteins. A common method for glycan analysis is to employ exoglycosidase cleavage to release N-linked glycans from glycoproteins or glycopeptides using Peptide-N-Glycosidase F (PNGase F). However, the glycan-protein linkages in bacteria are different and there is no enzyme available to release glycans from bacterial glycoproteins. In addition, free glycans have also been described in mammalian cells, bacteria, yeast, plants, and fish. In this article, we present a method that can characterize the N-linked glycosylation system in Campylobacter jejuni by detecting asparagine (Asn)-linked and free glycans that are not attached to their target proteins. In this method, total proteins from C. jejuni were digested by Pronase E with a higher enzyme to protein ratio (2:1&#8722;3:1) and a longer incubation time (48&#8722;72 h). The resulted Asn-glycans and free glycans were then purified using porous graphitic carbon cartridges, permethylated, and analyzed by mass spectrometry.

This study presents a method for glycomics analysis of glycoproteins by a combination of pronase E digestion, permethylation, and mass spectrometry analysis. This method is capable of analyzing all types of N-linked glycans, including bacterial N-glycans.

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the ...

<meta charset="utf8"></head><body>使用糖组学指导的糖蛋白质组学分析结直肠癌组织

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven glycomics and glycoproteomics have emerged as powerful approaches to analyze liberated glycans and intact glycopeptides, respectively, offering a deeper understanding of the heterogeneous glycoproteome in the tumor microenvironment (TME). This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology, which firstly employs porous graphitized carbon-LC-MS/MS-based glycomics to elucidate the glycan structures and their quantitative distribution in the glycome of tumor tissues, cell populations, or bodily fluids being investigated. This allows for a comparative glycomics analysis to identify altered glycosylation across patient groups, disease stages, or conditions, and, importantly, serves to enhance the downstream glycoproteomics analysis of the same sample(s) by creating a library of known glycan structures, thus reducing the data search time and the glycoprotein misidentification rate. Focusing on N-glycoproteome profiling, the sample preparation steps for the glycomics-guided glycoproteomics method are detailed in this protocol, and key aspects of the data collection and analysis are discussed. The glycomics-guided glycoproteomics method provides quantitative information on the glycoproteins present in the TME and their glycosylation sites, site occupancy, and site-specific glycan structures. Representative results are presented from formalin-fixed paraffin-embedded tumor tissues from colorectal cancer patients, demonstrating that the method is sensitive and compatible with tissue sections commonly found in biobanks. Glycomics-guided glycoproteomics, therefore, offers a comprehensive view into the heterogeneity and dynamics of the glycoproteome in complex TMEs, generating robust biochemical data required to better understand the glycobiology of cancers.

<meta charset="utf8"></head><body>糖组学引导的糖蛋白质组学有助于在复杂的肿瘤微环境中全面分析糖蛋白质组

This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology that offers comprehensive insights into the heterogeneous glycoproteome in complex tumor microenvironments required to better understand the glycobiology of cancers.

糖组学引导的糖蛋白质组学有助于在复杂的肿瘤微环境中全面分析糖蛋白质组 

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven ...

定量糖组学和蛋白质组学组合式净化策略

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鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

有机溶剂基蛋白质沉淀，在质谱分析之前实现强大的蛋白质组纯化

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

糖组学引导的糖蛋白质组学有助于在复杂的肿瘤微环境中全面分析糖蛋白质组

糖组学引导的糖蛋白质组学有助于在复杂的肿瘤微环境中全面分析糖蛋白质组

糖组学引导的糖蛋白质组学有助于在复杂的肿瘤微环境中全面分析糖蛋白质组