Facilitating the Chemistry of Fluorosequencing

促进荧光测序的化学发展

• 批准号: 10623602
• 负责人: Eric V. Anslyn
• 金额: $ 38.44万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10623602
• 关键词: Amino Acids; BODIPY; C-terminal; Chemicals; Chemistry; Collaborations; Databases; Diagnostic; Disease; Dyes; Fluorescence Resonance Energy Transfer; Generations; Genomics; Health; Individual; Label; Lysine; Methods; Methylation; Microscope; N-terminal; Nucleic acid sequencing; Peptide Sequence Determination; Peptides; Positioning Attribute; Post-Translational Protein Processing; Protein Analysis; Proteins; Proteomics; Reporting; Running; Sampling; Series; Techniques; Technology; Time; Ubiquitination; Work; base; design; fluorophore; imaging modality; improved; nanopore; protein aminoacid sequence; sequencing platform; single molecule

ABSTRACT While advances in nucleic acid sequencing to achieve parallel and single molecule analysis have been astounding, analogous techniques for peptide/protein sequencing are lacking. Tandem mass-spec analysis still requires millions of copies of a protein and is inherently serial rather than parallel. Thus, the current approaches being applied to parallel and single molecule protein analysis primarily focus on the use of modified nanopores. But their ability to differentiate subtle differences between individual amino acids (AAs), or strings of AAs, only works in isolated cases. To create a parallel single molecule sequencing platform for peptides/proteins, in collaboration with the Marcotte group, we devised a technique called fluorosequencing. In this technique, we label AAs with fluorophores, and on a TIRF 4-channel microscope the N-terminal AAs are removed using classic Edman degradation. As fluorophore labelled AAs are iteratively removed, both their identity and position are revealed, generating a partial-sequence. The partial-sequences are compared to a genomic database of all possible proteins, thereby revealing the proteins in the sample. Mixtures of millions of peptides are analyzed in parallel; single molecules at a time. Albeit the method is functional, there are several aspects that require improvement to generate a mature technology. One obstacle that we will overcome is the general lack of approaches that allow sequential and selectively labelling of multiple amino acids on the same peptide/protein with different tags, as well as differentiating the N-terminal and C-terminal residues from lysine and Glu/Asp AAs, respectively. We will also explore labeling four AA within the following set: Cys, Lys, Tyr, Typ, His, Ser, Thr, Glu/Asp, Arg, PSer, PThr, PTyr (P = phospho). Moreover, we feel that fluorosequencing can be readily extended to post-translational modifications (PTMs) such as mono, di, and trimethylated Lys, as well as ubiquitination. The sequential and selective labelling of four AAs and/or PTMs will involve conjugation handles carrying “click” partners within a set of what we refer to as “click:clack” pairs, wherein the “clack” partner will carry one of a set of four fluorophores appropriate for the four channels of our TIRF microscope. In some previous sequencing runs we have discovered efficient donor/acceptor FRET between the fluorophores, thereby making the donor intensity either weak or entirely invisible. To solve this problem, we will explore the use of designed fluorophores whose emission can be turned on and off by intramolecular conjugate additions which are controlled by varying the pH. In addition, because a series of common fluorophores, such as Cy and BODIPY dyes, do not survive the TFA treatment used in Edman degradation, we are developing a base-induced method for N-terminal chain-end sequencing. Importantly, while each thrust is focused upon use in fluorosequencing, the advances thereof are broadly applicable to other proteomic approaches (mass spec and nanopore), imaging methods, as well as the general chemical manipulations of peptides and proteins.

摘要 虽然实现平行和单分子分析的核酸测序的进展已经被证实， 令人震惊的是，缺乏类似的肽/蛋白质测序技术。串联质谱分析釜 需要数百万个蛋白质拷贝，并且本质上是串行的而不是并行的。因此，目前的方法 应用于平行和单分子蛋白质分析主要集中于使用修饰的纳米孔。 但它们区分单个氨基酸（AAs）或氨基酸串之间细微差异的能力， 在个别情况下有效。为了创建肽/蛋白质的平行单分子测序平台， 与Marcotte小组合作，我们设计了一种称为荧光测序的技术。在这项技术中，我们 用荧光团标记AA，并在TIRF 4通道显微镜上使用经典的 埃德曼降解。由于荧光团标记的AA被反复去除，因此它们的身份和位置都被确定。 显示，生成部分序列。将部分序列与所有人的基因组数据库进行比较。 可能的蛋白质，从而揭示样品中的蛋白质。数百万种肽的混合物被分析， 平行的;每次单个分子。虽然该方法是功能性的，但有几个方面需要 改进以产生成熟的技术。我们将克服的一个障碍是， 允许在相同肽/蛋白质上连续和选择性标记多个氨基酸的方法 用不同的标签，以及区分N-末端和C-末端残基与赖氨酸和Glu/Asp AA， 分别我们还将探索标记以下组内的四个AA：Cys、Lys、Tyr、Typ、His、Ser、Thr， Glu/Asp、Arg、PSer、PThr、PTyr（P =磷酸）。此外，我们认为荧光测序可以很容易地扩展到 翻译后修饰（PTM），如单、二和三甲基化的Lys，以及泛素化。的 四种AA和/或PTM的顺序和选择性标记将涉及携带“点击”的缀合柄。 一组我们称为“点击：咔嗒”对中的伙伴，其中“咔嗒”伙伴将携带一组中的一个 四个荧光团的四个通道适合我们的TIRF显微镜。在之前的测序中 我们已经发现了荧光团之间的有效供体/受体FRET，从而使供体 强度弱或完全不可见。为了解决这个问题，我们将探索使用设计的荧光团 其发射可以通过分子内共轭加成来打开和关闭， 此外，由于一系列常见的荧光团，如Cy和BODIPY染料，不能在pH下存活， TFA处理用于Edman降解，我们正在开发一种碱诱导的方法用于N-末端链端 测序重要的是，虽然每一个推力都集中在荧光测序中的使用上，但其进步是显而易见的。 广泛适用于其他蛋白质组学方法（质谱和纳米孔）、成像方法以及 肽和蛋白质的一般化学操作。