Depth of proteome issues: a yeast isotope-coded affinity tag reagent study.
Depth of proteome issues: a yeast isotope-coded affinity tag reagent study.
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蛋白质组问题的深度：酵母同位素编码的亲和标签试剂研究。
DOI：
10.1074/mcp.m300110-mcp200
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发表时间：
2004
期刊：
影响因子：
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通讯作者：
Martin,Stephen
中科院分区：
文献类型：
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作者：
Parker,KennethC;Patterson,Dale;Williamson,Brian;Marchese,Jason;Graber,Armin;He,Feng;Jacobson,Allan;Juhasz,Peter;Martin,Stephen
作为优化蛋白质组学实验的测试案例，我们选择了一个酵母模型系统，其中upf1基因（一种参与无义介导的mRNA衰变的蛋白质）通过同源重组被敲除。将5个完整的同位素编码亲和标签（ICAT）实验结果进行综合，其中2个采用基质辅助激光解吸/电离（MALDI）串联质谱法（MS/MS）， 3个采用电喷雾质谱法（MS/MS）。我们试图评估多肽鉴定的可重复性，并开发一个信息学结构，尽可能地表征鉴定过程，特别是关于脆弱的鉴定。ICAT试剂系统的可切割形式(Gygiet al.(1999)Nat。生物技术学报，17,994-999)进行定量。大多数蛋白的表达在upf1敲除后没有显著变化。正如预期的那样，Upf1蛋白本身被下调，而与精氨酸生物合成有关的蛋白的表达可重复地增加。最初，似乎大约有10%的蛋白质在表达水平上发生了变化，但在对数据进行更彻底的检查后，发现大多数这些明显的变化可以用重/轻对重叠引起的量化伪影来解释。总共鉴定了大约700种蛋白质，并进行了高可信度和定量分析。鉴定了许多具有化学修饰的肽，以及具有非典型色氨酸末端的肽。几乎所有这些修饰的肽都与最丰富的酵母蛋白相对应，而有些则被认为是低可信度的“单击”蛋白。为了提高我们对鉴定的信心，在MALDI实验中，多肽的亲本质量与附近的成分进行了校准。此外，除了最初使用的Mascot分数外，还为每个光谱收集了五个反映识别不同方面的新参数。讨论了这些评分参数与蛋白质鉴定置信度之间的相互关系。
As a test case for optimizing how to perform proteomics experiments, we chose a yeast model system in which theUPF1gene, a protein involved in nonsense-mediated mRNA decay, was knocked out by homologous recombination. The results from five complete isotope-coded affinity tag (ICAT) experiments were combined, two using matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) and three using electrospray MS/MS. We sought to assess the reproducibility of peptide identification and to develop an informatics structure that characterizes the identification process as well as possible, especially with regard to tenuous identifications. The cleavable form of the ICAT reagent system (Gygiet al.(1999)Nat. Biotechnol.17, 994–999) was used for quantification. Most proteins did not change significantly in expression as a consequence of theupf1knockout. As expected, the Upf1 protein itself was down-regulated, and there were reproducible increases in expression of proteins involved in arginine biosynthesis. Initially, it seemed that about 10% of the proteins had changed in expression level, but after more thorough examination of the data it turned out that most of these apparent changes could be explained by artifacts of quantification caused by overlapping heavy/light pairs. About 700 proteins altogether were identified with high confidence and quantified. Many peptides with chemical modifications were identified, as well as peptides with noncanonical tryptic termini. Nearly all of these modified peptides corresponded to the most abundant yeast proteins, and some would otherwise have been attributed to “single hit” proteins at low confidence. To improve our confidence in the identifications, in MALDI experiments, the parent masses for the peptides were calibrated against nearby components. In addition, five novel parameters reflecting different aspects of identification were collected for each spectrum in addition to the Mascot score that was originally used. The interrelationship between these scoring parameters and confidence in protein identification is discussed.