Facilitating the Chemistry of Fluorosequencing

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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.