Large-scale mass spectrometric detection of variant peptides resulting from nonsynonymous nucleotide differences.
Large-scale mass spectrometric detection of variant peptides resulting from nonsynonymous nucleotide differences.
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DOI：
10.1021/pr4009207
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发表时间：
2014-01-03
影响因子：
4.4
通讯作者：
Smith LM
中科院分区：
文献类型：
--
作者：
Sheynkman GM;Shortreed MR;Frey BL;Scalf M;Smith LM
每个个体在他们的基因组中携带数千个非同义单核苷酸变体(NsSNV)，每个变体对应于编码蛋白质中的一个单一氨基酸多态性(SAP)。为了研究转录后调控、差异等位基因表达和其他重要的生物学过程，能够在蛋白质水平上直接检测和量化这些变异是很重要的。然而，在标准的蛋白质组学分析中，由于它们不存在于用于质谱学搜索的通用数据库中，因此通常不能在标准蛋白质组分析中检测到这些变异多肽。在这里，我们扩展了之前的工作，这些工作演示了如何使用由样本匹配的RNA-Seq数据构建的定制SAP数据库。我们收集了Jurkat细胞系的深度覆盖RNA-Seq数据，编译了表达的nsSNV集，使用这些信息构建了定制的SAP数据库，并与从同一样本获得的深度覆盖猎枪MS数据进行了搜索。这种方法能够检测到映射到395个nsSNV的421个SAP多肽。我们将这些多肽与从包含所有已知nsSNV的大型通用搜索数据库(DBSNP)中鉴定的多肽进行比较，发现来自DBSNP的搜索的SAP多肽中超过70%不被RNA-Seq数据支持，因此可能是假阳性。接下来，我们通过利用多个蛋白酶消化来增加来自RNA-Seq衍生数据库的SAP覆盖率，从而将变异检测增加到映射到504个nsSNV位点的695个SAP多肽。这些检测到的SAP多肽对应于中到高丰度的转录本(每百万个转录本30+)。SAP多肽包括192对等位基因；对其中51对的两个等位基因的相对表达水平进行了评估，发现在所有情况下都是可比的。
Each individual carries thousands of non-synonymous single nucleotide variants (nsSNVs) in their genome, each corresponding to a single amino acid polymorphism (SAP) in the encoded proteins. It is important to be able to directly detect and quantify these variations at the protein level in order to study post-transcriptional regulation, differential allelic expression, and other important biological processes. However, such variant peptides are not generally detected in standard proteomic analyses, due to their absence from the generic databases that are employed for mass spectrometry searching. Here, we extend previous work that demonstrated the use of customized SAP databases constructed from sample-matched RNA-Seq data. We collected deep coverage RNA-Seq data from the Jurkat cell line, compiled the set of nsSNVs that are expressed, used this information to construct a customized SAP database, and searched it against deep coverage shotgun MS data obtained from the same sample. This approach enabled detection of 421 SAP peptides mapping to 395 nsSNVs. We compared these peptides to peptides identified from a large generic search database containing all known nsSNVs (dbSNP) and found that more than 70% of the SAP peptides from this dbSNP-derived search were not supported by the RNA-Seq data, and thus are likely false positives. Next, we increased the SAP coverage from the RNA-Seq derived database by utilizing multiple protease digestions, thereby increasing variant detection to 695 SAP peptides mapping to 504 nsSNV sites. These detected SAP peptides corresponded to moderate to high abundance transcripts (30+ transcripts per million, TPM). The SAP peptides included 192 allelic pairs; the relative expression levels of the two alleles were evaluated for 51 of those pairs, and found to be comparable in all cases.
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影响因子：
4.4
作者：
Bunger, Maureen K.;Cargile, Benjamin J.;Stephenson, James L., Jr.
通讯作者：
Stephenson, James L., Jr.
来自1,092个人基因组的遗传变异的综合图。
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
7
作者：
Harrow J;Frankish A;Gonzalez JM;Tapanari E;Diekhans M;Kokocinski F;Aken BL;Barrell D;Zadissa A;Searle S;Barnes I;Bignell A;Boychenko V;Hunt T;Kay M;Mukherjee G;Rajan J;Despacio-Reyes G;Saunders G;Steward C;Harte R;Lin M;Howald C;Tanzer A;Derrien T;Chrast J;Walters N;Balasubramanian S;Pei B;Tress M;Rodriguez JM;Ezkurdia I;van Baren J;Brent M;Haussler D;Kellis M;Valencia A;Reymond A;Gerstein M;Guigó R;Hubbard TJ
通讯作者：
Hubbard TJ
影响因子：
14.9
作者：
Hubbard, T;Barker, D;Clamp, M
通讯作者：
Clamp, M
影响因子：
0.8
作者：
Chernobrovkin, A. L.;Mitkevich, V. A.;Archakov, A. I.
通讯作者：
Archakov, A. I.