Chibuike Maximillian, Rathnayaka Chathurika, Shivanka Suresh, Choi Junseo, Verber Matthew, Park Sunggook, Soper Steven A
Department of Chemistry, The University of Kansas, Lawrence, Kansas 66045, United States.
Center of Biomodular Multiscale Systems for Precision Medicine, The University of Kansas, Lawrence, Kansas 66045, United States.
Anal Chem. 2025 Jan 14;97(1):427-435. doi: 10.1021/acs.analchem.4c04542. Epub 2024 Dec 23.
We are developing a unique protein identification method that consists of generating peptides proteolytically from a single protein molecule (i.e., peptide fingerprints) with peptide detection and identification carried out using nanoscale electrochromatography and label-free resistive pulse sensing (RPS). As a step in realizing this technology, we report herein the nanoscale electrochromatography of model peptides using thermoplastic columns with surfaces engineered to identify peptides via their molecularly dependent mobility (i.e., time-of-flight, ToF). ToFs were elucidated using a dual in-plane nanopore sensor, which consisted of two in-plane nanopores placed on either end of the nanoelectrochromatography column. The surface of the nanocolumn, which consisted of poly(methyl methacrylate) (PMMA), was activated with an O plasma, creating surface carboxylic acid groups (-COOH) inducing a surface charge on the column wall as well as affecting its hydrophilicity. To understand scaling effects, we carried out microchip and nanochannel electrochromatography of the peptides labeled with an ATTO 532 reporter to allow for single-molecule tracking. Our results indicated that the apparent mobilities of the model peptides did not allow for their separation in a microchannel, but when performed in a nanocolumn, clear differences in their apparent mobilities could be observed especially when operated at high electric field strengths. We next performed label-free detection of peptides using the dual in-plane nanopore sensor with the two pores separated by a 5 μm (length) column with a 50 nm width and depth. When a single peptide molecule passed through an in-plane nanopore, the sensor read a pair of resistive pulses with a time difference equivalent to ToF. We identified the peptides by evaluating their ToF, normalized RPS current transient amplitude (Δ/), and RPS peak dwell time (). We could identify the model peptides with nearly 100% classification accuracy at the single-molecule level using machine learning with a single molecule measurement requiring <10 ms.
我们正在开发一种独特的蛋白质鉴定方法，该方法包括从单个蛋白质分子中通过蛋白水解生成肽段（即肽指纹图谱），并使用纳米级电色谱和无标记电阻脉冲传感（RPS）进行肽段检测和鉴定。作为实现该技术的一个步骤，我们在此报告使用热塑性柱对模型肽进行纳米级电色谱分析，该柱的表面经过工程设计，可通过肽段的分子依赖性迁移率（即飞行时间，ToF）来鉴定肽段。使用双平面纳米孔传感器阐明了ToF，该传感器由放置在纳米电色谱柱两端的两个平面内纳米孔组成。由聚甲基丙烯酸甲酯（PMMA）组成的纳米柱表面用O等离子体活化，产生表面羧酸基团（-COOH），在柱壁上诱导表面电荷并影响其亲水性。为了理解缩放效应，我们对用ATTO 532报告分子标记的肽段进行了微芯片和纳米通道电色谱分析，以实现单分子跟踪。我们的结果表明，模型肽段的表观迁移率不允许它们在微通道中分离，但在纳米柱中进行时，可以观察到它们表观迁移率的明显差异，特别是在高电场强度下操作时。接下来，我们使用双平面纳米孔传感器对肽段进行无标记检测，两个孔由一个长度为5μm、宽度和深度为50nm的柱隔开。当单个肽分子通过平面内纳米孔时，传感器读取一对电阻脉冲，其时间差相当于ToF。我们通过评估肽段的ToF、归一化RPS电流瞬态幅度（Δ/）和RPS峰停留时间（）来鉴定肽段。使用机器学习，我们可以在单分子水平上以近100%的分类准确率鉴定模型肽段，单次分子测量所需时间小于10毫秒。
