Facilitating the Chemistry of Fluorosequencing

摘要 虽然实现并行和单分子分析的核酸测序的进展 令人震惊的，类似的多肽/蛋白质测序技术是缺乏的。串联式质谱计 需要数百万个蛋白质拷贝，而且本质上是串联的，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在修饰纳米孔的使用上。 但它们区分单个氨基酸(AA)或氨基酸串之间细微差异的能力仅 在个别情况下有效。为了创建一个并行的多肽/蛋白质单分子测序平台，在 通过与马科特团队的合作，我们设计了一种叫做荧光测序的技术。在这种技术中，我们 用荧光团标记AA，并在TIRF 4通道显微镜上使用CLASSIC去除N端AA 埃德曼降级。随着荧光团标记的AA被反复移除，它们的身份和位置都被 显示出来，生成一个部分序列。将部分序列与所有的基因组数据库进行比较 可能的蛋白质，从而揭示了样本中的蛋白质。数以百万计的多肽混合物在 平行的；一次只有一个分子。尽管该方法是实用的，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普遍缺乏 允许在同一多肽/蛋白质上顺序和选择性地标记多个氨基酸的方法 具有不同的标签，以及区分N-端和C-端残基与赖氨酸和Glu/Asp氨基酸， 分别进行了分析。我们还将探索标记以下集合中的四个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)，如一、二和三甲基化的赖氨酸，以及泛素化。这个 四个AA和/或PTM的顺序和选择性标记将涉及带有“点击”的接合手柄 在我们所说的“咔哒”对中的伙伴，其中“咔哒”伙伴将携带一组中的一个 适用于我们的TIRF显微镜的四个通道的四个荧光团。在之前的一些排序中 我们已经发现有效的供体/受体在荧光团之间产生焦虑感，从而使供体 强度很弱或完全看不见。为了解决这个问题，我们将探索使用设计的荧光团 它的发射可以通过分子内的共轭加成来开启和关闭，这些加成是由不同的 PH值。此外，由于一系列常见的荧光团，如Cy和BODIPY染料，不能在 TFA在Edman降解中的应用，我们正在开发一种碱基诱导N-末端链端的方法 测序。重要的是，虽然每个推力都集中在荧光测序中的使用上，但其进步是 广泛适用于其他蛋白质组学方法(质谱学和纳米孔)、成像方法以及 多肽和蛋白质的一般化学操作。

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.