A label-free mass spectrometry method for the quantification of protein isotypes.
A label-free mass spectrometry method for the quantification of protein isotypes.
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DOI：
10.1016/j.ab.2009.07.052
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发表时间：
2009-12-15
影响因子：
2.9
通讯作者：
Himes RH
中科院分区：
文献类型：
--
作者：
Winefield RD;Williams TD;Himes RH
成功的定量质谱学(MS)需要将质谱仪的响应与分析物丰度联系起来的策略，响应依赖于更多的因素，而不仅仅是分析物丰度。依赖于标记的策略依赖于将同位素标记的内标结合到样品中。目前的无标记策略(在没有内部标准的情况下执行)对于分析不适合同位素标记但不太准确的样品很有用。在这里，我们描述了一种适用于相关基因(同种类型)产物分析的无标记技术。这种方法使家族内不变的胰蛋白酶多肽序列可以作为内置的内部标准，而同型特异性多肽序列可以报告各种同型的数量。消除过程可靠地将胰酶释放的标准肽和报告肽从不可靠释放的肽中分离出来。这些报告多肽和标准多肽的特定MS响应因子可以使用合成多肽来确定。对HeLa微管蛋白酶切的分析表明，来自βI-、βII-、βIII-、βIVb-和βV-微管蛋白的多肽，其中8个是合适的，以及5个用于β-微管蛋白亚型定量的标准肽。为了说明这一方法的实用性，我们确定了βI-微管蛋白占总β微管蛋白的77%，βIII微管蛋白占总Hela微管蛋白的3.2%。
Successful quantitative mass spectrometry (MS) requires strategies to link the mass spectrometer response to the analyte abundance, with the response being dependent on more factors than just analyte abundance. Label-dependent strategies rely on the incorporation of an isotopically labeled internal standard into the sample. Current label-free strategies (performed without internal standards) are useful for analyzing samples that are unsuitable for isotopic labeling but are less accurate. Here we describe a label-free technique applicable to analysis of products from related genes (isotypes). This approach enables the invariant tryptic peptide sequences within the family to serve as “built-in” internal standards and the isotype-specific peptide sequences to report the amount of the various isotypes. A process of elimination segregates reliably trypsin-released standard and reporter peptides from unreliably released peptides. The specific MS response factors for these reporter and standard peptides can be determined using synthetic peptides. Analysis of HeLa tubulin digests revealed peptides from βI-, βII-, βIII-, βIVb-, and βV-tubulin, eight of which were suitable; along with five standard peptides for quantification of the β-tubulin isotypes. To show the utility of this method, we determined that βI-tubulin represented 77% and βIIItubulin represented 3.2% of the total HeLa β-tubulin.
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影响因子：
3.9
作者：
Alterman, MA;Kornilayev, B;Yakovlev, D
通讯作者：
Yakovlev, D
影响因子：
7.4
作者：
Hagman, Charlotte;Ricke, Darrell;Bitsch, Francis
通讯作者：
Bitsch, Francis
影响因子：
15
作者：
Pasa-Tolic, L;Jensen, PK;Smith, RD
通讯作者：
Smith, RD
影响因子：
4.8
作者：
Kamath, K;Wilson, L;Jordan, MA
通讯作者：
Jordan, MA
影响因子：
4.4
作者：
Khodiyar, Varsha K.;Maltais, Lois J.;Lovering, Ruth C.
通讯作者：
Lovering, Ruth C.