公开(公告)号：US20030054570A1

公开(公告)日：2003-03-20

申请号：US10044708

申请日：2001-10-22

申请人： Genetics Institute, Inc.

发明人： Yongchang Qiu ,  Jack H. Wang ,  Rodney M. Hewick

IPC分类号： G01N033/543

CPC分类号： G01N33/6848 ,  G01N33/6851 ,  G01N2458/15 ,  Y10T436/13 ,  Y10T436/24 ,  Y10T436/25125

摘要： Arginine-containing cysteine-modifying compounds useful for MALDI-MS analysis of proteins are provided. These compounds termed isotope-coded ionization enhancement reagents (ICIER) can provide ionization enhancement in MALDI-MS, relative quantitation, and additional database searching constraints at the same time without any extra sample manipulation. More specifically, ICIER increase the ionization efficiency of cysteine-containing peptides by attachment of a guanidino functional group. ICIER also increase the overall hydrophilicity of these peptides due to the hydrophilic nature of ICIER and thus increase the percentage of recovery of these peptides during sample handling and processing such as in-gel digestion or liquid chromatography. Finally, a combination of both light and heavy ICIER provides an accurate way to obtain relative quantitation of proteins by MALDI-MS and additional database searching constraints (number of cysteine residues in every single peptide peak) to increase the confidence of protein identification by peptide mass mapping.

摘要翻译： 提供了可用于蛋白质MALDI-MS分析的含精氨酸的半胱氨酸修饰化合物。 这些称为同位素编码电离增强试剂（ICIER）的化合物可以在MALDI-MS中提供电离增强，相对定量和额外的数据库搜索约束，同时没有任何额外的样品操作。 更具体地，ICIER通过连接胍基官能团来增加含半胱氨酸的肽的电离效率。 由于ICIER的亲水性，ICIER还增加了这些肽的整体亲水性，因此在样品处理和加工（如凝胶内消化或液相色谱）期间增加了这些肽的回收百分比。 最后，轻和重的ICIER的组合提供了通过MALDI-MS和附加的数据库搜索约束（每个肽峰中的半胱氨酸残基数）获得蛋白质的相对定量的精确方式，以增加肽质量的蛋白质鉴定的置信度 映射。

公开(公告)号：US20020164809A1

公开(公告)日：2002-11-07

申请号：US10045170

申请日：2001-10-22

申请人： Genetics Institute, Inc.

发明人： Yongchang Qiu ,  Jack H. Wang ,  Rodney M. Hewick

IPC分类号： G01N033/00

CPC分类号： G01N33/6848 ,  C07D405/12 ,  G01N30/461 ,  G01N30/463 ,  G01N30/468 ,  G01N30/7233 ,  G01N33/532 ,  G01N33/58 ,  G01N33/6803 ,  Y10T436/10 ,  Y10T436/105831 ,  Y10T436/13 ,  Y10T436/24 ,  Y10T436/25125 ,  B01D15/362 ,  B01D15/325

摘要： The method of the invention provides novel compounds, termed acid-labile isotope-coded extractants (ALICE), for quantitative mass spectrometric analysis of protein mixtures. The compounds contain a thiol-reactive group that is used to capture cysteine-containing peptides from all peptide mixtures, an acid-labile linker, and a non-biological polymer. One of the two acid-labile linkers is isotopically labeled and therefore enables the direct quantitation of peptides/proteins through mass spectrometric analysis. Because no functional proteins are required to capture peptides, a higher percentage of organic solvent can be used to solubilize the peptides, particularly hydrophobic peptides, through the binding, washing and eluting steps, thus permitting much better recovery of peptides. Moreover, since the peptides are covalently linked to the non-biological polymer (ALICE), more stringent washing is allowed in order to completely remove non-specifically bound species. Finally, peptides captured by ALICE are readily eluted from the polymer support under mild acidic condition with high yield and permit the direct down stream mass spectrometric analysis without any further sample manipulation. In combination with our novel dual column two dimensional liquid chromatography-mass spectrometry (2D-LC-MS/MS) design, the ALICE procedure proves to a general approach for quantitative mass spectrometric analysis of protein mixtures with better dynamic range and sensitivity.

摘要翻译： 本发明的方法提供了新的化合物，称为酸不稳定同位素编码的提取物（ALICE），用于蛋白质混合物的定量质谱分析。 该化合物含有硫醇反应性基团，其用于从所有肽混合物，酸不稳定接头和非生物聚合物捕获含半胱氨酸的肽。 两个酸不稳定接头之一是同位素标记的，因此能够通过质谱分析直接定量肽/蛋白质。 由于不需要功能性蛋白来捕获肽，所以可以使用较高百分比的有机溶剂来通过结合，洗涤和洗脱步骤来增溶肽，特别是疏水性肽，从而可以更好地回收肽。 此外，由于肽与非生物聚合物（ALICE）共价连接，所以允许更严格的洗涤以完全除去非特异性结合的物质。 最后，ALICE捕获的肽在温和的酸性条件下以高产率容易地从聚合物载体上洗脱，并允许直接的下游质谱分析，无需进一步的样品操作。 结合我们的双柱二维液相色谱 - 质谱（2D-LC-MS / MS）设计，ALICE方法证明了具有更好的动态范围和灵敏度的蛋白质混合物的定量质谱分析的一般方法。