Deep Proteome Coverage Based on Ribosome Profiling Aids Mass Spectrometry-based Protein and Peptide Discovery and Provides Evidence of Alternative Translation Products and Near-cognate Translation Initiation Events
Deep Proteome Coverage Based on Ribosome Profiling Aids Mass Spectrometry-based Protein and Peptide Discovery and Provides Evidence of Alternative Translation Products and Near-cognate Translation Initiation Events
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DOI：
10.1074/mcp.m113.027540
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发表时间：
2013-07-01
影响因子：
7
通讯作者：
Van Damme, Petra
中科院分区：
文献类型：
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作者：
Menschaert, Gerben;Van Criekinge, Wim;Van Damme, Petra
越来越多的研究涉及基因和蛋白质表达数据的综合分析，利用新技术，如下一代转录组测序和高灵敏度质谱（MS）仪器。最近，已经描述了一种称为核糖体分析（或RIBO-seq）的策略，该策略基于核糖体保护的mRNA片段的深度测序，间接监测蛋白质合成。我们设计了一种蛋白质基因组学方法，构建了一个自定义的蛋白质序列搜索空间，由Swiss-Prot和RIBO-seq衍生的翻译产物构建，适用于MS/MS光谱鉴定。为了记录使用构建的深度蛋白质组数据库的影响，我们进行了两种替代的基于MS的蛋白质组学策略，如下所示：（i）常规鸟枪蛋白质组学和（ii）N-末端组合分数对角色谱（COFRADIC）方法。虽然前一种技术给出了蛋白质和肽水平的总体评估，但后一种技术特别能够分离N-末端肽，非常适合验证RIBO-seq衍生的（替代）翻译起始位点谱。我们证明，这种蛋白质基因组学方法提高了2.5%的整体蛋白质鉴定率（e。G.新的蛋白质产物、新的蛋白质剪接变体、单核苷酸多态性变体蛋白质和已知蛋白质的N-末端延伸形式）。此外，使用该定制数据库，除了鉴定4个翻译的上游ORF之外，N-末端COFRADIC数据的鉴定导致检测到16个替代起始位点，其产生N-末端延伸的蛋白变体。值得注意的是，这些新的翻译产物的表征揭示了多个近同源（非AUG）起始密码子的使用。随着深度测序技术变得越来越标准，越来越便宜和广泛，我们预计mRNA测序，特别是定制的RIBO-seq将成为基于MS的蛋白质或肽鉴定过程中不可或缺的。基础的质谱蛋白质组学数据已存入ProteomeXchange Consortium，数据集标识符为PXD 000124。
An increasing number of studies involve integrative analysis of gene and protein expression data, taking advantage of new technologies such as next-generation transcriptome sequencing and highly sensitive mass spectrometry (MS) instrumentation. Recently, a strategy, termed ribosome profiling (or RIBO-seq), based on deep sequencing of ribosome-protected mRNA fragments, indirectly monitoring protein synthesis, has been described. We devised a proteogenomic approach constructing a custom protein sequence search space, built from both Swiss-Prot-and RIBO-seq-derived translation products, applicable for MS/MS spectrum identification. To record the impact of using the constructed deep proteome database, we performed two alternative MS-based proteomic strategies as follows: (i) a regular shotgun proteomic and (ii) an N-terminal combined fractional diagonal chromatography (COFRADIC) approach. Although the former technique gives an overall assessment on the protein and peptide level, the latter technique, specifically enabling the isolation of N-terminal peptides, is very appropriate in validating the RIBO-seq-derived (alternative) translation initiation site profile. We demonstrate that this proteogenomic approach increases the overall protein identification rate 2.5% (e. g. new protein products, new protein splice variants, single nucleotide polymorphism variant proteins, and N-terminally extended forms of known proteins) as compared with only searching UniProtKB-SwissProt. Furthermore, using this custom database, identification of N-terminal COFRADIC data resulted in detection of 16 alternative start sites giving rise to N-terminally extended protein variants besides the identification of four translated upstream ORFs. Notably, the characterization of these new translation products revealed the use of multiple near-cognate (non-AUG) start codons. As deep sequencing techniques are becoming more standard, less expensive, and widespread, we anticipate that mRNA sequencing and especially custom-tailored RIBO-seq will become indispensable in the MS-based protein or peptide identification process. The underlying mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000124.