Credit: NIST

来源：NIST

NEW YORK – While technical hurdles remain, nanopore-based protein analysis is approaching feasibility as scientists have recently demonstrated potential methods for tackling the challenge.

纽约——虽然技术障碍仍然存在，但基于纳米孔的蛋白质分析正在接近可行性，因为科学家最近展示了应对这一挑战的潜在方法。

Both academic and industry-affiliated research groups have published on techniques for moving full-length proteins through nanopores in a controlled manner and making identifications based on the resulting signal, and at least one company, Portal Biotech, has begun to place nanopore-based protein analyzers in the hands of early adopters..

学术和行业附属研究小组都发表了关于以受控方式将全长蛋白质通过纳米孔并根据产生的信号进行鉴定的技术，至少有一家公司门户生物技术公司已经开始将基于纳米孔的蛋白质分析仪置于早期采用者手中。。

Nanopore biosensing typically uses the changes in electric current produced as a molecule translocates through the pore to determine its identity. To date, this approach has mainly been used for sequencing nucleic acids — most prominently by Oxford, UK-based Oxford Nanopore Technologies — but researchers have also been pursuing it for protein analysis..

纳米孔生物传感通常使用分子通过孔转移时产生的电流变化来确定其身份。迄今为止，这种方法主要用于核酸测序-最主要的是位于英国牛津的牛津纳米孔技术公司-但研究人员也一直在寻求将其用于蛋白质分析。。

Compared to nucleic acid sequencing, nanopore-based protein analysis is significantly more difficult. Researchers must contend with 20 amino acids when analyzing proteins versus just four bases in DNA, as well as a multitude of post-translational modifications. They also face the challenge of having to unfold proteins prior to passing them through the pores with enough control and consistency to generate reproducible signals for protein identification..

与核酸测序相比，基于纳米孔的蛋白质分析要困难得多。研究人员在分析蛋白质时必须处理20个氨基酸，而DNA中只有4个碱基，以及大量的翻译后修饰。他们还面临着挑战，即必须在蛋白质通过孔之前展开蛋白质，并具有足够的控制和一致性，以产生可重复的蛋白质识别信号。。

The upside of nanopore-based protein measurements could be substantial, as it could enable detailed analyses of full-length proteins with single-molecule sensitivity, something not currently possible using established proteomic technologies like mass spectrometry or immunoassays.

基于纳米孔的蛋白质测量的优势可能是巨大的，因为它可以对具有单分子敏感性的全长蛋白质进行详细分析，这是目前使用质谱或免疫分析等既定蛋白质组学技术所不可能的。

Perhaps most immediate on the horizon is nanopore-based protein fingerprinting, in which nanopores are used not to read an amino acid sequence but to generate a 'fingerprint” signal that is characteristic of a protein. Researchers can use this fingerprint to identify the protein as well as use deviations from the expected signal to identify potential variations in its sequence or posttranslational modifications..

也许最直接的是基于纳米孔的蛋白质指纹图谱，其中纳米孔不是用来读取氨基酸序列，而是用来产生蛋白质特有的“指纹”信号。研究人员可以使用这种指纹识别蛋白质，也可以使用与预期信号的偏差来识别其序列或翻译后修饰的潜在变异。。

True de novo sequencing, in which researchers read each amino acid in a protein as it passes through a nanopore, is likely a longer way off, with some in the field still questioning whether this will ultimately be possible. Some also debate whether it will prove necessary or if fingerprinting approaches will be sufficient..

真正的从头测序（研究人员在蛋白质通过纳米孔时读取蛋白质中的每个氨基酸）可能还有更长的路要走，该领域的一些人仍在质疑这最终是否可能。一些人还争论这是否有必要，或者指纹识别方法是否足够。。

The translocation challenge

易位挑战

For either application, an effective strategy for moving target proteins through a pore is key. Broadly speaking, such strategies fall into two categories — enzymatic, in which enzymes like DNA helicases or the protein unfoldase ClpX are used to translocate proteins, and non-enzymatic, in which physical forces such as electrophoresis or electro-osmotic flow are used to translocate target proteins..

对于任何一种应用，通过孔移动靶蛋白的有效策略都是关键。从广义上讲，这些策略分为两类-酶促，其中DNA解旋酶或蛋白质解折叠酶ClpX等酶用于转运蛋白质，非酶促，其中电泳或电渗流等物理力用于转运目标蛋白质。。

In a study published last year in Nature Nanotechnology, a team led by Hagan Bayley, professor of chemical biology at the University of Oxford and one of the founders of Oxford Nanopore, described the use of electro-osmosis for nanopore protein analysis. The process uses nanopores selective for particular ions in a liquid.

在去年发表在《自然纳米技术》上的一项研究中，牛津大学化学生物学教授、牛津纳米孔创始人之一哈根·贝利（Hagan Bayley）领导的团队描述了电渗技术在纳米孔蛋白质分析中的应用。该过程使用对液体中特定离子具有选择性的纳米孔。

When a potential is applied to the nanopore, it pulls those ions toward it, which carry water molecules with them. This flow of liquid can be used to unfold proteins and move them through the nanopore..

当电位被施加到纳米孔上时，它会将那些携带水分子的离子拉向纳米孔。这种液体流动可用于展开蛋白质并将其通过纳米孔。。

Bayley said that he and his colleagues view electro-osmosis as 'the most promising method” for protein translocation, noting that the enzymatic tools used for nanopore-based DNA sequencing are not necessarily well suited to protein analysis. A non-enzymatic approach could also be simpler than an enzyme-based one, as it would require less manipulation of the target proteins..

Bayley说，他和他的同事认为电渗是蛋白质转运的“最有前途的方法”，并指出用于基于纳米孔的DNA测序的酶工具不一定非常适合蛋白质分析。非酶方法也可能比基于酶的方法更简单，因为它需要较少的靶蛋白操作。。

Aleksei Aksimentiev, a physicist at the University of Illinois at Urbana-Champaign whose work includes research into nanopore-based biosensing, highlighted demonstrations by Bayley's lab and others of the use of electro-osmotic force as an important recent advance for the field. Prior to this work, he said, it was unclear whether this force would be sufficient to move proteins through nanopores.

伊利诺伊大学厄本那-香槟分校的物理学家Aleksei Aksimentiev的工作包括基于纳米孔的生物传感研究，他强调了Bayley实验室和其他实验室使用电渗透力的证明，这是该领域最近的一个重要进展。他说，在这项工作之前，尚不清楚这种力是否足以使蛋白质通过纳米孔。

'It looks like it is,” he said. 'And that is a really great development.”.

“看起来是这样，”他说这真的是一个很大的发展。”。

Aksimentiev noted that there are difficulties to the electro-osmotic approach, particularly around controlling the consistency of protein movement through the pore, which may vary across the length of the pore and depend on the charge of the protein sequence passing through the pore at a given time.

Aksimentiev指出，电渗方法存在困难，特别是在控制蛋白质通过孔运动的一致性方面，这可能在孔的长度上有所不同，并且取决于在给定时间通过孔的蛋白质序列的电荷。

Enzymatic approaches could provide more consistent translocation, he said, though electro-osmotic force alone could prove sufficient for fingerprinting applications..

他说，酶促方法可以提供更一致的易位，尽管仅电渗透力就足以用于指纹识别应用。。

On the enzymatic side, Oxford Nanopore and a team including Aksimentiev and Delft University of Technology researcher Cees Dekker have separately explored strategies that link DNA to target proteins and then use DNA helicases to pull the protein-DNA complex through a nanopore. One potential limitation to this approach, however, is read length.

在酶学方面，牛津纳米孔和包括阿克西门提夫（Aksimentiev）和代尔夫特理工大学（Delft University of Technology）研究员塞斯·德克尔（Cees Dekker）在内的团队分别探索了将DNA与靶蛋白连接起来，然后使用DNA解旋酶将蛋白质-DNA复合物拉过纳米孔的策略。然而，这种方法的一个潜在限制是读取长度。

Xiuqi Chen, a postdoctoral researcher in Dekker's lab, said that he and his colleagues are currently able to look at peptides in the range of 20 to 30 amino acids using the technique..

。。

Additionally, several researchers are pursuing methods that combine enzymatic and non-enzymatic approaches. This month, a team of researchers led by Jeff Nivala, a molecular engineering professor at the University of Washington, published a study in Nature in which they used electrophoresis to pull proteins through a nanopore and then ClpX to pull it back out of the pore in a slower, more reproducible manner, generating better signals for identifying the protein and detecting amino acid variants and PTMs..

此外，一些研究人员正在寻求结合酶和非酶方法的方法。本月，由华盛顿大学分子工程教授杰夫·尼瓦拉（Jeff Nivala）领导的一组研究人员在《自然》杂志上发表了一项研究，他们使用电泳将蛋白质拉过纳米孔，然后使用ClpX以较慢，更可重复的方式将其拉出孔，从而产生更好的信号来识别蛋白质并检测氨基酸变体和PTM。。

Nivala said that while researchers are still exploring various methods for translocating proteins through nanopores, he believes some sort of enzymatic motor will be key. 'I just don't think [you have] the reproducibility, the control of how the strand moves if you don't have a motor,” he said. 'It's just too variable, in my opinion.”.

尼瓦拉说，虽然研究人员仍在探索各种通过纳米孔转运蛋白质的方法，但他相信某种酶促马达将是关键他说：“我只是认为，如果你没有电机，你就没有重现性，无法控制钢绞线的运动。”在我看来，它太多变了。”。

Nanopore protein analysis firm Portal Biotech is also taking a combined approach, pairing electro-osmotic force with a nanopore engineered to include a proteosome to unfold and translocate target proteins. In 2021, Giovanni Maglia, the company's cofounder and CSO as well as a professor of chemical biology at Groningen University, published a paper in Nature Chemistry on a nanopore-proteosome complex his lab had engineered and used for protein measurements.

。2021年，该公司的联合创始人、CSO以及格罗宁根大学化学生物学教授乔瓦尼·马格里亚（GiovanniMaglia）在《自然化学》杂志上发表了一篇论文，介绍了他实验室设计并用于蛋白质测量的纳米孔蛋白体复合物。

In 2023, he and his colleagues published a study in Nature Biotechnology on the use of electro-osmotic flow for unfolding and translocating proteins..

。。

The combination of these forces allows the company to pass proteins through its nanopores with 'almost metronomic precision,” said Andrew Heron, Portal Biotech cofounder and CEO. Heron was previously senior director of advanced research at Oxford Nanopore.

Portal Biotech联合创始人兼首席执行官安德鲁·赫伦（AndrewHeron）说，这些力量的结合使该公司能够以“几乎节律的精确度”将蛋白质通过其纳米孔。Heron曾任牛津纳米孔高级研究主任。

Near-term potential

近期潜力

With these advances in controlling unfolding and translocation, the field has drawn closer to being able to make meaningful protein measurements. Currently, these are fingerprinting measurements and are typically made in a targeted fashion on small sets of proteins or peptides, often either purified or synthetically generated..

随着控制展开和易位的这些进展，该领域已经接近能够进行有意义的蛋白质测量。目前，这些是指纹测量，通常以有针对性的方式对少量蛋白质或肽进行测量，这些蛋白质或肽通常是纯化的或合成的。。

Nanopore-based protein fingerprinting can potentially provide information that is difficult to generate using approaches like mass spec, said Chen. He cited as an example more in-depth analysis of known proteins to allow for the identification and localization of multiple PTMs or other alterations.

陈说，基于纳米孔的蛋白质指纹图谱可能会提供难以使用质谱等方法生成的信息。他举了一个例子，对已知蛋白质进行更深入的分析，以便识别和定位多个PTM或其他改变。

With mass spec, 'when you have multiple sites of modification, it can be a problem to identify which amino acids are being modified, but with [nanopores] we know the peptide is going through the pore in a certain way, and the signal is different at certain steps, so we can pretty reliably say which amino acids are modified.”.

通过质谱分析，“当你有多个修饰位点时，识别哪些氨基酸被修饰可能是一个问题，但是对于[纳米孔]，我们知道肽以某种方式穿过孔，并且信号在某些步骤是不同的，因此我们可以非常可靠地说哪些氨基酸被修饰。”。

In a recent study published in the Journal of the American Chemical Society, Bayley and colleagues used chemical binders (phos-tags) specific to protein phosphorylation to further distinguish the signal produced by these modifications, allowing them to detect phosphorylation along a polypeptide chain of more than 700 amino acids..

在最近发表在《美国化学学会杂志》上的一项研究中，Bayley及其同事使用了特定于蛋白质磷酸化的化学粘合剂（phos标签），以进一步区分这些修饰产生的信号，从而使他们能够检测700多个氨基酸的多肽链上的磷酸化。。

Bayley noted, however, that 'technical improvements” are still needed. 'If a 1,000 amino acid polypeptide goes through the pore and is phosphorylated at, say, seven positions, do we see all seven?” he said. 'Right now, the answer is ‘not always.' And so that has to be improved.”

然而，贝利指出，仍然需要“技术改进”他说现在，答案是“不总是”因此必须改进。”

Nivala said that based on recently and soon-to-be published studies, nanopore-based protein fingerprinting 'is probably here” for applications in which researchers are looking at a well-defined set of protein targets.

尼瓦拉说，根据最近和即将发表的研究，基于纳米孔的蛋白质指纹图谱“可能已经出现”，用于研究人员正在寻找一组明确定义的蛋白质靶标的应用。

He offered as an example an experiment in which, 'I'm looking at this particular peptide or this particular protein, and I want to know, does it have this particular modification at this site?”

他举了一个例子，在这个实验中，“我正在研究这种特殊的肽或这种特殊的蛋白质，我想知道，它在这个位点是否有这种特殊的修饰？”

Nivala said he expects this will be expanded to broader fingerprinting efforts in the near term, in which experimentally generated nanopore data can be compared to an empirically built database of nanopore data for specific proteins. The next step, he said, could be to generate databases of simulated nanopore data, in which the signal produced by a given protein could be predicted by its sequence and identifications could be made by matching experimentally generated nanopore signal to the predicted signals, much as is done in discovery mass spec..

尼瓦拉说，他预计这将在短期内扩展到更广泛的指纹识别工作，其中可以将实验生成的纳米孔数据与经验构建的特定蛋白质纳米孔数据数据库进行比较。他说，下一步可能是生成模拟纳米孔数据的数据库，其中给定蛋白质产生的信号可以通过其序列进行预测，并且可以通过将实验产生的纳米孔信号与预测信号进行匹配来进行鉴定，就像发现质谱中所做的那样。。

Nivala suggested that, initially, methods for proteome-scale fingerprinting will likely be built on such an approach. He noted that in their recent Nature paper, he and his colleagues demonstrated that they could predict nanopore signals based on an amino acid sequence that matched a protein's actual experimental signal..

。他指出，在最近的《自然》论文中，他和他的同事证明了他们可以根据与蛋白质实际实验信号匹配的氨基酸序列预测纳米孔信号。。

Portal Biotech is working along similar lines. Heron said the company 'routinely measures proteins that are more than 100 kilodaltons in size.” He noted that one advantage of measuring such large proteins, as opposed to shorter peptides, is that their signature is highly unique, making for easier identification..

门户生物技术公司（Portal Biotech）也在沿着类似的路线工作。Heron说，该公司“通常会测量大小超过100千道尔顿的蛋白质。”他指出，与较短的肽相比，测量如此大的蛋白质的一个优点是它们的特征非常独特，便于识别。。

'In the mass spec space, even though it is very accurate, if you have a peptide, it is still quite ambiguous what molecule it came from. But if you feed a 50 or 100 kilodalton protein [through a nanopore], it becomes totally unambiguous what the molecule is because you've got so much data,” Heron said..

“在质谱空间中，即使它非常准确，如果你有一种肽，它来自什么分子仍然是相当模糊的。但是，如果你[通过纳米孔]喂养一个50或100千道尔顿的蛋白质，那么这个分子是什么就变得完全明确了，因为你有这么多的数据，”Heron说。。

Portal Biotech, which was founded in 2021, has been using its experimental nanopore data to train machine-learning algorithms to predict nanopore signals from protein sequences with the goal of producing databases of simulated signals that can be used to make identifications. Heron said the company is now at the point where it is capable of producing such simulated databases and identifying individual proteins out of thousands of potential candidates with accuracies above 90 percent, though it has not yet published peer-reviewed research detailing this work..

成立于2021年的门户生物技术公司（Portal Biotech）一直在使用其实验性纳米孔数据来训练机器学习算法，以预测蛋白质序列中的纳米孔信号，目的是生成可用于识别的模拟信号数据库。Heron表示，该公司目前能够生产此类模拟数据库，并从数千种潜在候选蛋白质中识别出单个蛋白质，准确率在90%以上，尽管该公司尚未发表详细介绍这项工作的同行评审研究。。

The road to de novo sequencing

从头测序之路

True de novo sequencing of proteins is a harder problem. Identifying each of 20 amino acids as they pass through a nanopore along with PTMs of interest is a daunting challenge. Adding to the challenge is the fact that as a protein translocates, multiple amino acids are interacting with the nanopore at any moment in time, creating highly complex signals that are difficult to deconvolute..

蛋白质的真正从头测序是一个更难的问题。鉴定20个氨基酸中的每一个以及感兴趣的PTM通过纳米孔是一项艰巨的挑战。更大的挑战是，随着蛋白质易位，多种氨基酸随时与纳米孔相互作用，产生难以解卷积的高度复杂的信号。。

'You have 20 amino acids, and then we suspect that around eight amino acids typically contribute to a given signal,” Chen said. 'You're in the billions of parameters … so the complexity could be very formidable.” He added that it was unclear to him if a supervised machine-learning algorithm could ever deal with that level of complexity..

“你有20个氨基酸，然后我们怀疑大约8个氨基酸通常对给定的信号有贡献，”陈说您拥有数十亿个参数……因此复杂性可能非常可怕。”他补充说，他不清楚有监督的机器学习算法是否能够处理这种复杂程度。。

Chen suggested that one possible way to address this issue could be to reduce the complexity of potential amino acid combinations by, for instance, using features like the different charge states of different amino acids to narrow down the possible amino acids present, thus reducing the size of the combinatorial space..

陈建议，解决这个问题的一种可能的方法是降低潜在氨基酸组合的复杂性，例如，使用不同氨基酸的不同电荷态等特征来缩小可能存在的氨基酸，从而减小组合空间的大小。。

Nivala said that while he believes de novo sequencing will ultimately prove possible, innovation on the chemistry side, and in nanopore design in particular, will be necessary. He suggested that shallower pores that contain fewer amino acids at a time could be key to simplifying the signal enough to enable sequencing-based approaches..

尼瓦拉说，虽然他相信从头测序最终将被证明是可能的，但在化学方面，特别是在纳米孔设计方面的创新将是必要的。他认为，一次含有较少氨基酸的较浅毛孔可能是简化信号以实现基于测序的方法的关键。。

Aksimentiev likewise said that nanopores designed to confine a smaller number of amino acids — perhaps around two or three — would be key to enabling de novo sequencing. Another possible option, he said, would be to sever amino acids one at a time from target proteins and feed them singly through the nanopore.

。他说，另一种可能的选择是从目标蛋白质中一次切断一个氨基酸，然后将它们单独通过纳米孔喂养。

'The problem there is that you have to capture all of them and capture them in sequence,” he said. 'That is a technical challenge.”.

他说：“问题是，你必须捕获所有这些目标，并按顺序捕获它们。”。

Heron expressed optimism that the field would ultimately achieve de novo sequencing, saying that improvements in nanopore technology will be needed but that 'the majority of the work will be on training and building better and better machine learning.”

Heron对该领域最终将实现从头测序表示乐观，他表示需要改进纳米孔技术，但“大部分工作将用于培训和构建越来越好的机器学习。”

He cited the example of nanopore-based DNA sequencing, which he noted has seen dramatic advances in capabilities since the field's initial efforts. 'We think we'll walk a similar pathway with proteins,” he said. 'It's very hard to say how long that pathway is because it just requires time, people, effort, and money.” .

他引用了基于纳米孔的DNA测序的例子，他指出，自该领域的初步努力以来，该领域的能力取得了巨大进步。”他说：“我们认为我们将在蛋白质方面走类似的道路。”很难说这条路有多长，因为它只需要时间、人员、努力和金钱。”。

Commercial prospects

商业前景

Nanopore-based protein analysis has to date remained largely an academic enterprise, with commercial entrants having thus far failed to bring products to market.

迄今为止，基于纳米孔的蛋白质分析在很大程度上仍然是一家学术企业，商业进入者迄今未能将产品推向市场。

French firm DreamPore launched in 2017 with the goal of developing a nanopore protein analysis system, though the company now appears to be inactive and did not return requests for comment.

法国DreamPore公司于2017年成立，目标是开发纳米孔蛋白质分析系统，尽管该公司目前似乎不活跃，没有回复置评请求。

In 2018, Dekker and his Delft colleague Chirlmin Joo launched a company called Bluemics to commercialize nanopore-based protein detection technology developed in their labs, but they shut down the company after struggling to find investors.

2018年，Dekker和他的代尔夫特同事Chirlmin Joo成立了一家名为Bluemics的公司，将实验室开发的基于纳米孔的蛋白质检测技术商业化，但由于难以找到投资者，他们关闭了该公司。

Portal Biotech appears to be off to a more promising start. According to Heron, the company, which raised $11 million in venture funding in 2022, has begun placing instruments with early adopters ranging from academic labs to small and large biopharma companies, though he declined to name any specific firms.

门户生物技术似乎有了一个更有希望的开始。据Heron称，该公司于2022年筹集了1100万美元的风险投资，已开始向早期采用者提供仪器，从学术实验室到小型和大型生物制药公司，尽管他拒绝透露任何具体公司的名称。

The company is also analyzing samples sent by outside customers, Heron said. 'It's still at the stage where we continue to refine the software and capabilities, but the most important thing is for us to put it in the hands of our customers, for them to tell us how we can improve it further for their particular applications.” .

Heron说，该公司还在分析外部客户发送的样本我们仍处于不断改进软件和功能的阶段，但最重要的是我们将其交给客户，让他们告诉我们如何进一步改进其特定应用程序。”。

Oxford Nanopore also continues to work on applying its technologies to protein analysis. In an email, Lakmal Jayasinghe, senior VP of R&D biologics at the company, said it has 'made considerable progress toward this goal,” though he added that it is still working 'to establish methods to prepare full-length proteins that can be threaded through the nanopore in a uniform fashion” and that 'amino [acid] calling is particularly challenging.” He added that while the company's ultimate aim is de novo sequencing, it also intends 'to develop application-specific products and methods to enable protein fingerprinting.”.

牛津纳米孔公司还继续致力于将其技术应用于蛋白质分析。该公司研发生物制品高级副总裁拉马尔·贾亚辛格（Lakmal Jayasinghe）在一封电子邮件中表示，该公司“在实现这一目标方面取得了相当大的进展”，但他补充说，该公司仍在努力“建立制备全长蛋白质的方法，这些蛋白质可以以统一的方式穿过纳米孔”，而“氨基酸的调用尤其具有挑战性”他补充说，虽然该公司的最终目标是从头测序，但它还打算“开发特定于应用程序的产品和方法，以实现蛋白质指纹识别。”。

Some of Nivala's work, including that presented in the recent Nature study, is partially funded by Oxford Nanopore. In the study, he and his colleagues used the company's MinIon platform for their measurements.

Nivala的一些工作，包括最近自然研究中提出的工作，部分由牛津纳米孔资助。在这项研究中，他和他的同事使用该公司的MinIon平台进行测量。

As with other entrants in the single-molecule protein analysis space, like Quantum-Si and Nautilus Biotechnology, nanopores will likely first be used for narrowly targeted applications.

与量子硅和鹦鹉螺生物技术等单分子蛋白质分析领域的其他进入者一样，纳米孔可能首先用于狭义靶向应用。

Initially, 'we will be going into the targeted proteomic space,” Heron said. 'A protein or a limited library of proteins [of interest] is very accessible for us.” He said the company plans at first to focus on the drug development space, facilitating, for instance, in-depth characterization of drug biomarkers or targets or, in the case of biologics, the drugs themselves..

最初，“我们将进入目标蛋白质组学领域，”Heron说蛋白质或有限的蛋白质库（感兴趣的）对我们来说是非常容易获得的。”他说，该公司首先计划专注于药物开发空间，例如促进药物生物标志物或靶标的深入表征，或者在生物制剂的情况下，药物本身。。

Longer term, though, Heron said he expects the technology will be useful for 'deep discovery” proteomics. His cofounder Maglia echoed this sentiment. 'There are complexities of course, but I think there is no intrinsic limitation,” he said. 'With time, you can tackle these problems.”

然而，从长远来看，Heron说，他预计这项技术将对“深度发现”蛋白质组学有用。他的联合创始人Maglia也表达了这种观点当然有复杂性，但我认为没有内在的局限性，”他说随着时间的推移，你可以解决这些问题。”