Name: lerlic-ms/ms for in-depth characterization and quantification of glutamine and asparagine deamidation in shotgun proteomics
Uploaded: 2017-04-09T15:00:00.000+00:00
Duration: 8 min 1 s
Description: Watch this Scientific Journal Video about LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics at JoVE.com

，新加坡（-OF-NMRC IRG-0003-2016），和NTU-NHG老化研究资助的国家医学研究理事会（:这项工作是在部分地由教育的新加坡教育部（授予ARC9 / 15级2）资助项目格兰特ARG / 14017）。我们想表达我们的感谢和最诚挚的感谢安德鲁·阿尔珀特博士和PolyLC团队亲切与成为可能这项研究包装材料提供给我们。

1.包装长的长度阴离子交换（LAX）毛细管柱 （注意:虽然LAX列可以在家里装，因为我们在这个协议描述，LAX列也可购得，见材料与试剂的表的其他细节）。 <ol><li>暂停50毫克弱阴离子交换包装材料的在3.5mL的包装缓冲器 （ 表1）以制备淤浆。 </li><li>组装该毛细管柱（50厘米长 - 200微米内径（ID）管）的端部使用一个母对阴配合，一个套圈和阴螺母。放置屏幕1/16&quot; 1微米OD的孔尺寸的女性与阴配件内（注意:此处所用的屏幕必须具有更小的孔尺寸比包装材料的颗粒尺寸，以防止来自所述材料的泄漏柱）。 </li><li>包用在4500 PSI操作压力泵浆毛细管。 （注:打包coluMN，直到包装材料是在列的条目）可见。 </li><li>如在点1.2描述组装该毛细管柱的另一端。 </li></ol> 2.样品制备该协议概述LERLIC-MS / MS的应用程序来分析人类脑组织作为模型蛋白质组。 （注意:如果使用其它组织或蛋白质组样品中，样品制备程序应适应。） <ol><li>组织均匀化退火: 一个。洗涤脑组织（50〜100毫克）与五分钟三次1×磷酸盐缓冲溶液。 湾均质化的组织在1:1:2.5的组织/金属珠/ SDC匀浆缓冲液 （ 表2）比（W / W / V），在4℃下，在安全锁定管以最大强度使用组织匀浆5分钟。 （注:如前面在3所示SDC匀浆缓冲液可以包括蛋白酶抑制剂）中 C。离心组织匀浆，获得来回M中的前一步骤，以10,000×克，4℃10分钟，并收集上清液到新的1.5mL管中。 天。重复BC对于剩余的粒料的步骤和直到没有沉淀观察到结合上清液作为根据需要多次。 （注:如果您有重复步骤BC两倍以上，样品和珠子移动到新的安全锁管以防止在离心步骤样品的损失）。 即量化通过二喹啉甲酸测定（BCA）12所得到的匀浆的蛋白质浓度。 </li><li>脱氧胆酸钠（SDC）辅助脑匀浆的溶液内胰蛋白酶消化13。 一个。添加10mM的二硫苏糖醇（DTT）（使用表3中的解决方案5所示的原液）到所获得的匀浆以发起的蛋白质二硫键键的还原的最终浓度。 湾在浴中30分钟期间孵育匀浆预先设定为60℃。 C。添加到匀浆使用表4的解决方案7所示的储备溶液的20mM碘乙酰胺（IAA）使终浓度。 天。孵育45分钟期间含有IAA在黑暗中在室温下将组织匀浆。 即稀释脑匀浆用稀释缓冲液的两倍（ 表3的解决方案6）。 F。在37℃下30分钟期间孵育的第二还原步骤的样品。 G。添加溶解在表2中的解决方案2的测序级修饰胰蛋白酶在蛋白质与酶比率1:50（重量/重量）。 H。通过温育过夜，在30℃消化用胰蛋白酶匀浆。 一世。通过添加甲酸（FA）的0.5％最终浓度淬灭第二天酶消化。 （注:FA的加入将导致在酸性条件下SDC盐沉淀）。 学家轻轻涡旋含有SDC precipi样品大老。 ķ。通过在12,000×g下，在10分钟期间4℃离心该样品沉淀下来SDC盐。 湖收集上清液和液体转移到一个干净新管中。 （注:从SDC颗粒这一步骤的移液时小心不要再中止和/或收集盐。） 米重新溶解在SDC再溶解缓冲液 （ 表5）的SDC粒料下剧烈涡旋1分钟。 ñ。重复步骤JL两次以恢复沉淀的肽，并结合上清。 （注:参见Serra 等 2016 13上的进一步细节的SDC-协助溶液胰蛋白酶消化协议这里适配。） </li><li>胰蛋白酶的消化脱盐样品。 一个。执行使用1克C-18盒式磁带消化的样品的脱盐。 （注意:使用一个大体积盒（5毫升）的独立地蛋白得到保证的样品中剩余的SDC的盐的适当的清洁的量之前到LC-MS / MS注射）。 湾用5mL的乙腈（ACN）执行1克C-18盒式磁带的调理。 C。由空调1克C-18盒式磁带通过5mL的清理缓冲液 （ 表6），以除去任何残留的有机溶剂。 天。加载样品到1克C-18盒。 即执行3 - 5清理步骤，使用5毫升清理缓冲器的每个步骤中的1克C-18柱的样品。 F。洗脱使用5毫升的洗脱缓冲液 （ 表7）的1克C-18盒式磁带的脱盐肽。 G。在真空中干燥浓缩洗脱的肽。 C。重新构成干燥的样品中的洗脱缓冲液的200μL的终体积。 （注意:使用剧烈和长（&gt; 10分钟），涡旋，随后在超声浴超声处理之后30分钟期间以完全重新悬浮在注射缓冲液的干燥肽） 天。调整样品LERLIC-MS / MS对ANA的注射体积lyze蛋白1 3之间，以微克。 </li></ol> 3.一维LERLIC-MS / MS分离<ol><li>液相色谱条件: 流动相: A:0.1％甲酸的水（ 表8）。 B:0.1％甲酸的乙腈（ 表9） 流量:0.4微升/分钟 柱:LAX毛细管柱（50厘米长 - 为200μmID） 一个。使用下面的1200分钟梯度:95％B 40分钟，95 - 434分钟85％B，85 - 为522分钟70％B，70 - 为124分钟，35％B，35 - 3％B 45分钟，等度在5分钟的3％B，3 - 95％B历时7分钟，并保持等度在95％B 23分钟。 湾执行使用LAX毛细管如塞拉＆盖勒特-帕劳等人指出的肽的分离。使用超高压液相色谱3。 </li><li>质谱仪配置: 一个。配置扫描事件的详细信息进行数据采集交替betwe烯全傅立叶变换质谱（FT-MS）和傅里叶变换 - 串联质谱法（FT-MS / MS）使用以下参数: A.1。数据采集​​模式:正 A.2 FT-MS参数: 质量范围:350 - 2000 M / Z 分辨率60000 微扫描:1每光谱 自动增益控制:1×10 6 A.3 FT-MS / MS参数: 分段模式:高能碰撞解离（HCD） A.4质量范围:150 - 2000 M / Z A.5分辨率:30,000 A.6前N个离子:10 微扫描:1每光谱 充电状态:&gt; 2+ 隔离宽度:2达 A.7自动增益控制:1×10 6 湾执行如使用配备有纳米电喷雾离子源在1.5千伏工作轨道阱质谱仪在3所示的肽的质谱检测。 </li></ol> 4.数据分析<ol><li>执行prote在数据库检索到LERLIC-MS / MS使用获得使用特定的蛋白质组软件中的以下参数的数据:（注:进一步的细节请参见第3）。 一个。离子公差为10ppm 湾碎片离子公差:0.05沓 C。假发现率（FDR）:1％ 天。数据库:UniProt的人体数据库 即修正修改:Carbamydomethylation在半胱氨酸 F。可变修饰（如果需要由软件）:氧化（MET），脱酰胺（Asn和谷氨酰胺）。 </li><li>自信表征和异构体脱酰胺产物在LERLIC-MS / MS数据量化: 一个。从LERLIC-MS / MS数据库导出搜索结果的spreedsheet软件进行分析。 湾查找并提取脱酰胺肽及其非脱酰胺化的同行的整个列表。 C。使用所获得的肽的列表作为参考，以5ppm质量公差的提取使用一个适当的软件这些肽的离子色谱图（XICs）。 （注意:对用于XICs提取软件的详细信息，请参阅3）。 天。目测检查得到的XICs以识别所指示3的异构体产物的分离式双峰洗脱。 （注:在MS / MS鉴定的水平可以容易地发现通过在数据库中检索结果的两个不同的保留时间的存在被引导的那些肽的异构体产物。） 即从每个脱酰胺位点/肽的异构体产物的相对定量具有基于在所获得的XICs每个识别出的异构体的峰面积进行。 </li></ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+effect+of+trypsin+digestion+buffers+on+artificial+deamidation.">Evaluation of the effect of trypsin digestion buffers on artificial deamidation.</a> J Proteome Res. 14 (2), 1308-1314 (2015).</li><li>Liu, S., Moulton, K. R., Auclair, J. R., Zhou, Z. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mildly+acidic+conditions+eliminate+deamidation+artifact+during+proteolysis:+digestion+with+endoprotease+Glu-C+at+pH+4.5.">Mildly acidic conditions eliminate deamidation artifact during proteolysis: digestion with endoprotease Glu-C at pH 4.5.</a> Amino Acids. 48 (4), 1059-1067 (2016).</li></ol>

作者什么都没有透露。

在该视频文章，我们提出LERLIC-MS / MS 3的步骤一步协议，一种方法来进行深入的表征和准确地确定蛋白质脱酰胺和涉及在此蛋白质修饰的酶过程的程度。 LERLIC-MS / MS是基于静电排斥亲水性相互作用色谱（ERLIC）27的原则下使用的长长度（50厘米）的LAX。使用长长度列，如在我们的研究3中所示，根据它们的等电点27 ERLIC的电位最大...

脱酰胺是一种蛋白质翻译后修饰（PTM），通过天冬酰胺（ASN）和/或谷氨酰胺（Gln）残基1的变形例引入了一个负电荷的蛋白质骨架。 1比2:此修改而影响的Asn残基处的共同3生成异构产物异天冬氨酸（异Asp）和正 -天冬氨酸（ASP）。尽管如此，该比率可以通过修复酶L-isoaspartyl甲基（PIMT）3，4的干预而改变。类似地，谷氨酰胺残基的脱酰胺化，生成以预期1异构γ-谷氨酸（γ-Glu），并且的α-谷氨酸亚型（α-GLU）:7的比例3,5，但此比例可通过的作用而移普遍存在的酶的转谷氨酰胺酶2和其它转谷氨酰胺酶，转谷氨酰胺酶包括1，一个电子nzyme最近被确定为与大脑中的6外囊泡。 脱酰胺的起源可以是自发的或酶促的，前者是特别常见的谷氨酰胺残基，其中的转谷氨酰胺酶和其它酶通过转酰氨调解帧间/分子内交联（参见第3对谷氨酰胺酰氨进一步细节及其含义在几种慢性和致命人类疾病）。因此，脱酰胺是具有在所述结构上的一个关键反响和功能的影响分子4，7，8，并且需要进行了深入的化学表征3中的其不同的生物化学后果的光包括其服务老化9的作为分子时钟PTM 。 虽然天冬酰胺残留的脱酰胺已<html>相对充分表征通过自下而上鸟枪蛋白质组学1,10，谷氨酰胺残基的脱酰胺化仍然没有超出由基于电子的自由基碎片11模型化合物的具有挑战性的分析的合适表征方法。我们最近开发了一种新型一维鸟枪蛋白质组学策略（LERLIC-MS / MS）3，使在一次分析中从复杂生物样品和模型化合物谷氨酰胺和天冬酰胺脱酰胺同种型的分离。 LERLIC-MS / MS是基于使用的长长度（50厘米）离子交换柱（LAX）上静电排斥亲水性相互作用色谱（ERLIC）模式下工作的胰蛋白酶消化的肽的分离和耦合到串联质谱（LC- MS / MS）。这个新的分析策略已被用于表征和比较定量每个脱酰胺化残基在人脑组织中的程度F"&gt; 3。然而，这里概述的协议将提供旨在研究蛋白质脱酰胺的特殊性在感兴趣的生化上下文LERLIC-MS / MS的视频成像。 道德守则 本协议的所有程序已通过在新加坡南洋理工大学的机构审查委员会，并根据该机构准则已执行。

<table><tbody><tr><td>PolyCAT 3 &amp;micro;m 100-&amp;Aring; (bulk material)</td><td>PolyLC Inc.</td><td>Special order</td><td></td></tr><tr><td>Long-length ion exchange capillary column 50 cm - 200 &amp;micro;m ID</td><td>PolyLC Inc.</td><td>Special order</td><td></td></tr><tr><td>PEEKsil Tubing 1/16&quot; OD x 200 &amp;micro;m ID x 50 cm length</td><td>SGE Analytical Science under Trajan Scientific Australia&amp;nbsp;</td><td>620050</td><td></td></tr><tr><td>Female-to-female fitting for 1/16&quot; OD tubbing</td><td>Upchurch Scientific</td><td>UPCHF-125</td><td></td></tr><tr><td>Female nut for microferule</td><td>Upchurch Scientific</td><td>UPCHP-416</td><td></td></tr><tr><td>Microferule</td><td>Upchurch Scientific</td><td>UPCHF-132</td><td></td></tr><tr><td>Pressure Bomb NanoBaume</td><td>Western Fluids Engineering</td><td>SP-400</td><td></td></tr><tr><td>Shimadzu Prominence UFLC system</td><td>Shimadzu</td><td>Prominence UFLC</td><td></td></tr><tr><td>Bullet Blender</td><td>Next Advance</td><td>BBX24</td><td></td></tr><tr><td>Safe-lock tubes</td><td>Eppendorf&amp;nbsp;</td><td>T9661-1000EA</td><td></td></tr><tr><td>Stainless steel beads. 0.9 &amp;ndash; 2.0 mm. 1 lb. Non-sterile.</td><td>Next Advance</td><td>SSB14B</td><td></td></tr><tr><td>Table-top centrifuge&amp;nbsp;</td><td>Hettich Zentrifugen</td><td>Rotina 380 R</td><td></td></tr><tr><td>Standard Digital Heated Circulating Bath, 120VAC</td><td>PolyScience 8006 6L</td><td>8006A11B</td><td></td></tr><tr><td>Sep-pack c18 desalting cartridge 50 mg</td><td>Waters</td><td>WAT020805</td><td></td></tr><tr><td>Vacumm concentrator</td><td>Eppendorf&amp;nbsp;</td><td>Concentrator Plus System</td><td></td></tr><tr><td>Dionex UltiMate 3000 UHPLC&amp;nbsp;</td><td>Dionex</td><td>UltiMate 3000 UHPLC&amp;nbsp;</td><td></td></tr><tr><td>Orbitrap Elite mass spectrometer</td><td>Thermo Fisher Scientific Inc.</td><td>ORBITRAP ELITE</td><td></td></tr><tr><td>Michrom Thermo CaptiveSpray&amp;nbsp;</td><td>Michrom-Bruker Inc.</td><td>TCSI-SS2</td><td></td></tr><tr><td>Incubator INCUCELL&amp;nbsp;</td><td>MMM Group</td><td>INCUCELL111</td><td></td></tr><tr><td>Sequencing-grade modified trypsin</td><td>Promega</td><td>V5111</td><td></td></tr><tr><td>Protease inhibitor cocktail tablets</td><td>Roche</td><td>11836170001 (ROCHE)</td><td></td></tr><tr><td>Phosphate buffer solution 10x (diluted to 1x)</td><td>Sigma-Aldrich</td><td>P5493</td><td></td></tr><tr><td>Ammonium acetate</td><td>Sigma-Aldrich</td><td>A1542</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich</td><td>D6750</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich</td><td>D0632</td><td></td></tr><tr><td>Iodoacetamide</td><td>Sigma-Aldrich</td><td>i6125</td><td></td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich</td><td>F0507 (HONEYWELL)</td><td></td></tr><tr><td>Ammonium hydroxide</td><td>Sigma-Aldrich</td><td>338818 (HONEYWELL)</td><td></td></tr><tr><td>Acetonitrile HPLC grade</td><td>Sigma-Aldrich</td><td>675415</td><td></td></tr><tr><td>Isopropanol HPLC grade</td><td>Sigma-Aldrich</td><td>675431</td><td></td></tr><tr><td>Water HPLC grade</td><td>Sigma-Aldrich</td><td>14263</td><td></td></tr></tbody></table>

lerlic-ms/ms for in-depth characterization and quantification of glutamine and asparagine deamidation in shotgun proteomics

蛋白质脱酰胺化的表征是势在必行破译在人体病理学和其他生化上下文该蛋白的翻译后修饰（PTM）的作用（S）和潜力。为了进行蛋白质脱酰胺的表征，我们最近开发了一种新型长条静电排斥亲水性相互作用色谱 - 串联质谱（LERLIC-MS / MS）方法，其可以分离谷氨酰胺（Gln）和天冬酰胺（ASN）同种型脱酰胺的产物从模型化合物到高度复杂的生物样品。 LERLIC-MS / MS，因此，第一鸟枪蛋白质组学策略的Gln脱酰胺同种型的分离和定量。我们还证明，作为新颖性，在这里列出的样品处理方案稳定琥珀酰亚胺中间体允许由LERLIC-MS / MS表征。如图所示这个视频文章中LERLIC-MS / MS的应用可以帮助阐明目前不明蛋白质脱酰胺的n个分子阵列。此外，LERLIC-MS / MS提供了涵盖不同的生物学背景脱酰胺酶反应的进一步理解。

谷氨酰胺和天冬酰胺残基的脱酰胺化被认为在几种慢性和致命疾病14牵连退行性蛋白修饰（DPM）。已经证明，这种PTM可以预测的抗体和其他分子的半衰期和降解状态在人体内和相似的生物学背景1，15。蛋白质脱酰胺的意义，其实超越了生物医学的背景下，因此这种修改存在于不同的蛋白质组<sup...

Watch this Scientific Journal Video about LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics at JoVE.com

LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics

这里，我们提出的长条静电排斥亲水相互作用色谱 - 串联质谱（LERLIC-MS / MS）的方法的一个步骤一步协议。这是一种新的方法，使通过鸟枪蛋白质组学的谷氨酰胺和天冬酰胺脱酰胺亚型的第一次定量和定性。

LERLIC-MS / MS进行深入表征和谷氨酰胺的定量和天冬酰胺脱酰胺在鸟枪蛋白质组学

Characterization of protein deamidation is imperative to decipher the role(s) and potentialities of this protein posttranslational modification (PTM) in ...

lerlic-msms-for-depth-characterization-quantification-glutamine

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

8.1K 次观看.  Nanyang Technological University. 这里，我们提出的长条静电排斥亲水相互作用色谱 - 串联质谱（LERLIC-MS / MS）的方法的一个步骤一步协议。这是一种新的方法，使通过鸟枪蛋白质组学的谷氨酰胺和天冬酰胺脱酰胺亚型的第一次定量和定性。 

视频: LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics

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 ...

科研

化学

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

生物化学

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) ...

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.

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 Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

基于质谱的蛋白质组学方法用于全局和高置信度的蛋白质R-甲基化分析

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

Laser Microdissection-Based Protocol for the LC-MS/MS Analysis of the Proteomic Profile of Neuromelanin Granules

Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in dopaminergic neurons of the substantia nigra pars compacta. Besides neuromelanin, NMGs contain a variety of proteins, lipids, and metals. Although NMGs-containing dopaminergic neurons are preferentially lost in neurodegenerative diseases like Parkinson&#39;s disease and dementia with Lewy bodies, only little is known about the mechanism of NMG formation and the role of NMGs in health and disease. Thus, further research on the molecular characterization of NMGs is essential. Unfortunately, standard protocols for the isolation of proteins are based on density gradient ultracentrifugation and therefore require high amounts of human tissue. Thus, an automated laser microdissection (LMD)-based protocol is established here which allows the collection of NMGs and surrounding substantia nigra (SN) tissue using minimal amounts of tissue in an unbiased, automatized way. Excised samples are subsequently analyzed by mass spectrometry to decipher their proteomic composition. With this workflow, 2,079 proteins were identified of which 514 proteins were exclusively identified in NMGs and 181 in SN. The present results have been compared with a previous study using a similar LMD-based approach reaching an overlap of 87.6% for both proteomes, verifying the applicability of the revised and optimized protocol presented here. To validate current findings, proteins of interest were analyzed by targeted mass spectrometry, e.g., parallel reaction monitoring (PRM)-experiments.

A robust protocol is presented here for isolating neuromelanin granules from human post-mortem substantia nigra pars compacta tissue via laser microdissection. This revised and optimized protocol massively minimizes the required time for sample collection, reduces the required sample amount, and enhances the identification and quantification of proteins by LC-MS/MS analysis.

基于激光显微切割的协议，用于神经黑色素颗粒蛋白质组学谱的LC-MS / MS分析

Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in dopaminergic neurons of the substantia nigra pars compacta. ...

神经科学

Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to human brain function, as neurons are postmitotic and rely heavily on the autophagy-lysosome pathway to maintain cellular homeostasis. Despite advancements in the understanding of various lysosomal functions, capturing the highly dynamic communications between lysosomes and other cellular components is technically challenging, particularly in a high-throughput fashion. Here, a detailed protocol is provided for the recently published endogenous (knock-in) lysosome proximity labeling proteomic method in human induced pluripotent stem cell (hiPSC)-derived neurons. 
Both lysosomal membrane proteins and proteins surrounding lysosomes within a 10-20 nm radius can be confidently identified and accurately quantified in live human neurons. Each step of the protocol is described in detail, i.e., hiPSC-neuron culture, proximity labeling, neuron harvest, fluorescence microscopy, biotinylated protein enrichment, protein digestion, LC-MS analysis, and data analysis. In summary, this unique endogenous lysosomal proximity labeling proteomics method provides a high-throughput and robust analytical tool to study the highly dynamic lysosomal activities in live human neurons.

A neuronal lysosome proximity labeling proteomics protocol is described here to characterize the dynamic lysosomal microenvironment in human induced pluripotent stem cell-derived neurons. Lysosomal membrane proteins and proteins that interact with lysosomes (stably or transiently) can be accurately quantified in this method with excellent intracellular spatial resolution in live human neurons.

具有邻近标记蛋白质组学的神经元溶酶体相互作用组表征

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

选择反应监测质谱法进行绝对定量蛋白质

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

生物学

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

用数据无关采集质谱法定量测定点特异蛋白赖氨酸乙酰化和琥珀酰化化学计量学

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

Comprehensive Workflow of Mass Spectrometry-based Shotgun Proteomics of Tissue Samples

Recent advances in mass spectrometry have resulted in deep proteomic&#160;analysis along with the generation of&#160;robust and&#160;reproducible datasets. However, despite the considerable technical advancements, sample preparation from biospecimens such as patient blood, CSF, and tissue still poses considerable challenges. For identifying biomarkers, tissue proteomics often provides an attractive sample source to translate the research findings from the bench to the clinic. It can reveal potential candidate biomarkers for early diagnosis of cancer and neurodegenerative diseases such as Alzheimer&#39;s disease, Parkinson&#39;s disease, etc. Tissue proteomics also yields a wealth of systemic information based on the abundance of proteins and helps to address interesting biological questions.
Quantitative proteomics analysis can be grouped into two broad categories: a label-based and a label-free approach. In the label-based approach, proteins or peptides are labeled using stable isotopes such as SILAC (stable isotope labeling with amino acids in cell culture) or by chemical tags such as ICAT (isotope-coded affinity tags), TMT (tandem mass tag) or iTRAQ (isobaric tag for relative and absolute quantitation). Label-based approaches have the advantage of more accurate quantitation of proteins and using isobaric labels, multiple samples can be analyzed in a single experiment. The label-free approach provides a cost-effective alternative to label-based approaches. Hundreds of patient samples belonging to a particular cohort can be analyzed and compared with other cohorts based on clinical features. Here, we have described an optimized quantitative proteomics workflow for tissue samples using label-free and label-based proteome profiling methods, which is crucial for applications in life sciences, especially biomarker discovery-based projects.

The described protocol provides an optimized quantitative proteomics analysis of tissue samples using two approaches: label-based and label free quantitation. Label-based approaches have the advantage of more accurate quantitation of proteins, while a label-free approach is more cost-effective and used to analyze hundreds of samples of a cohort.