A theoretical justification for single molecule peptide sequencing.
A theoretical justification for single molecule peptide sequencing.
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DOI：
10.1371/journal.pcbi.1004080
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发表时间：
2015-02
影响因子：
4.3
通讯作者：
Marcotte EM
中科院分区：
文献类型：
--
作者：
Swaminathan J;Boulgakov AA;Marcotte EM
细胞、组织和生物体的蛋白质组反映了活跃的细胞过程，并响应细胞内和细胞外的线索而不断变化。蛋白质组的深度定量分析，特别是如果与mRNA和代谢物测量相结合，应该提供前所未有的细胞状态视图，更好地揭示细胞组分的功能和相互作用。分子诊断和生物标志物的发现应该特别受益于蛋白质组的准确定量，因为癌症等复杂疾病会改变蛋白质的丰度和修饰。目前，鸟枪质谱是高通量蛋白质鉴定和定量的主要技术;虽然功能强大，但缺乏高灵敏度和覆盖范围。我们绘制平行与下一代DNA测序，并提出了一种策略，称为荧光测序，测序肽在一个复杂的蛋白质样品在单分子的水平。在所提出的方法中，数百万个单独的荧光标记的肽被并行可视化，监测荧光强度的变化模式，因为N-末端氨基酸被顺序去除，并使用产生的荧光特征（荧光序列）来唯一地识别单个肽。我们介绍了荧光测序的理论基础，并通过使用Monte Carlo计算机模拟，我们探索其可行性，预测最可能的实验误差，量化其潜在的影响，并讨论了广泛的潜在效用提供了高通量肽测序技术。下一代DNA和RNA测序方法的发展已经改变了生物学，目前的平台每次运行产生超过10亿个测序读数。不幸的是，没有类似的规模和通量的方法存在，以确定和定量复杂的混合物中的特定蛋白质，在许多生物化学和分子诊断分析的关键瓶颈。迫切需要的是一种类似于下一代DNA测序的大规模并行方法，用于识别和定量样品中的肽或蛋白质。原则上，单分子肽测序可以实现这一目标，允许对数十亿个不同的肽进行平行测序，从而识别组成样品的蛋白质，并通过直接计数肽对其进行数字定量。在这里，我们讨论了单分子肽测序的理论考虑，提出了一种可能的实验策略，并使用计算机模拟来表征这种未来蛋白质组学技术的潜在实用性和不寻常的特性。
The proteomes of cells, tissues, and organisms reflect active cellular processes and change continuously in response to intracellular and extracellular cues. Deep, quantitative profiling of the proteome, especially if combined with mRNA and metabolite measurements, should provide an unprecedented view of cell state, better revealing functions and interactions of cell components. Molecular diagnostics and biomarker discovery should benefit particularly from the accurate quantification of proteomes, since complex diseases like cancer change protein abundances and modifications. Currently, shotgun mass spectrometry is the primary technology for high-throughput protein identification and quantification; while powerful, it lacks high sensitivity and coverage. We draw parallels with next-generation DNA sequencing and propose a strategy, termed fluorosequencing, for sequencing peptides in a complex protein sample at the level of single molecules. In the proposed approach, millions of individual fluorescently labeled peptides are visualized in parallel, monitoring changing patterns of fluorescence intensity as N-terminal amino acids are sequentially removed, and using the resulting fluorescence signatures (fluorosequences) to uniquely identify individual peptides. We introduce a theoretical foundation for fluorosequencing and, by using Monte Carlo computer simulations, we explore its feasibility, anticipate the most likely experimental errors, quantify their potential impact, and discuss the broad potential utility offered by a high-throughput peptide sequencing technology. The development of next-generation DNA and RNA sequencing methods has transformed biology, with current platforms generating >1 billion sequencing reads per run. Unfortunately, no method of similar scale and throughput exists to identify and quantify specific proteins in complex mixtures, representing a critical bottleneck in many biochemical and molecular diagnostic assays. What is urgently needed is a massively parallel method, akin to next-gen DNA sequencing, for identifying and quantifying peptides or proteins in a sample. In principle, single-molecule peptide sequencing could achieve this goal, allowing billions of distinct peptides to be sequenced in parallel and thereby identifying proteins composing the sample and digitally quantifying them by direct counting of peptides. Here, we discuss theoretical considerations of single molecule peptide sequencing, suggest one possible experimental strategy, and, using computer simulations, characterize the potential utility and unusual properties of this future proteomics technology.
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影响因子：
9.9
作者：
通讯作者：
--
影响因子：
3.4
作者：
Aitken, Colin Echeverria;Marshall, R. Andrew;Puglisi, Joseph D.
通讯作者：
Puglisi, Joseph D.
影响因子：
15
作者：
MacBeath, G;Koehler, AN;Schreiber, SL
通讯作者：
Schreiber, SL
影响因子：
46.9
作者：
Hoebe, R. A.;Van Oven, C. H.;Manders, E. M. M.
通讯作者：
Manders, E. M. M.
DOI：
10.1073/pnas.251525598
发表时间：
2001-12-04
影响因子：
11.1
作者：
Garcia-Parajo, MF;Koopman, M;van Hulst, NF
通讯作者：
van Hulst, NF