analysis of the microfluidics coupled mass photometry data to investigate low-affinity protein-protein interactions

使用微流体扩展质量亮度分析浓度范围

Protein-protein interactions underline most cellular functions, from immune regulation to DNA replication and translation. As a result, there is a fundamental need throughout the life sciences to investigate a vast range of interactions across diverse heterogenous complexes that are commonly formed. However, their detection, characterization, and quantification are often challenging, particularly for low-affinity interactions1.
Immunoprecipitation assays are often used to detect high-affinity interactions, but for low-affinity and transient interactions, detection is largely unfeasible2. Fluorescence techniques can also be used but require the potentially disruptive addition of fluorescent labels2. Cryo-EM can provide a structural snapshot and an ensemble readout of the protein complexes formed with high spatial resolution but also typically requires working at concentrations that are too low for imaging low-affinity interactions. Cryo-EM also brings challenges related to cost, accessibility, sample preparation, and analysis time3.
Additionally, surface plasmon resonance (SPR) has become a popular way to quantify protein-protein interactions, although it requires protein immobilization, which can affect the binding equilibrium and result in variable on-rates, thus reducing measurement accuracy4,5. It also involves several assay steps prior to data collection and analysis6.
Mass photometry is a single-molecule technique that has been used to analyze protein-protein interactions5,6,7. It works by measuring the mass of single molecules or complexes based on the light they scatter when they land on the surface of a glass coverslip8. Mass photometry measurements have been used to quantify binding affinities from the relative abundance of binding partners and the complexes they form5. Nevertheless, like other single-molecule techniques, the concentration of the sample to be measured should typically be less than 100 nM. If the concentration is higher, the molecules landing on the glass surface will overlap spatially, resulting in poor data quality7. Consequently, weaker interactions (KD ~micromolars), which dissociate at these lower concentrations, cannot be measured reliably since it is not possible to observe the necessary mixture of unbound and bound species5.
Here, we describe an approach that overcomes this limitation based on a new coupled microfluidics mass photometry device. Specifically, a microfluidics system is used in combination with the mass photometer to effectively expand the range of interactions that can be quantified by mass photometry. Microfluidics has been shown to offer a range of possibilities for investigating protein-protein interactions, including rapid dilution to detect weak interactions1,9. The system described herein operates by rapidly diluting the sample up to 10,000-fold on a microfluidic chip and immediately flowing it across the observation area of the chip, enabling the mass photometry measurement to start within 50 ms from when the molecules began the dilution process10. The dilution occurs when the sample and buffer are combined in a reverse Tesla valve mixer on the chip, with the relative flow rates of the two solutions determining the amount of dilution that occurs (see Protocol step 8). The flow rate is controllable with the microfluidic control software. Altering the flow rate can change the relative population of the species as it can impact the number of landing events on the surface of the glass, which is what is measured by the mass photometer.
The speed of the process is fast enough for the measurement to be completed before the integrity of the interaction has been disrupted (for further details, see also the Discussion). This can be understood through a brief look at the theory of first-order reactions, where <img alt="Equation 1" src="/files/ftp_upload/65772/65772eq01.jpg" style="margin: auto;" />. The forward (association) rate constant is kf, the backward (dissociation) rate constant is kb, and the equilibrium dissociation constant (KD) is defined as
KD = kb / kf
For protein binding, kf is generally limited by the reactants&#39; diffusion11 and so is restricted to the range of 106-107 M-1&#183;s-1. Because the range of is limited, a low-affinity (KD ~micromolars) reaction will have kb &#8776; 1 s-1. That is, kb = kf&#160;&#183; KD = (106 M-1&#183;s-1) (10-6 M) = 1 s-1, with the complex&#39;s half-life around 0.7 s11,12.
Our example system is the binding of the IgG monoclonal antibody trastuzumab to the soluble domain of the IgG neonatal Fc receptor (FcRn), which are known interacting partners13. Previously published data obtained using conventional mass photometry alone (i.e., with manual dilution of samples) showed that the proteins form multiple species. FcRn monomers, FcRn dimers, and unbound IgG were clearly visible, while IgG-FcRn complexes (at 1:1 and 1:2 ratios) were also detected (at pH 5.0) but only with very low abundance5. This observation prompts the question of whether IgG-FcRn complex formation could be more clearly detected if measured at a higher concentration. Indeed, combining mass photometry with a coupled rapid dilution approach described here provided more robust evidence of complex formation by an increase in their measured particles.
The mass photometry and microfluidics protocol described here makes it possible to characterize the formation of complexes with a KD up to the micromolar range. An empirical determination of the KD will require further improvements on the flow sensor accuracy, pump stability, chip-to-chip variations, and measurement location inside the observation window, as all these factors would influence the time from when the sample is diluted to being measured.
The same approach could be applied to investigate binding among any soluble proteins, provided they have distinct molecular weights (separated by at least 25 kDa) that fall into the range suitable for analysis with a mass photometer (30 kDa to 6 MDa). The insights obtained could be helpful for studies in a range of contexts - from gaining a mechanistic understanding of cellular functions to the design of new biotherapeutic drugs.

作者聚焦：使用 MassFluidix 技术表征溶液中低亲和力蛋白质相互作用

通过将微流体与质量光度法相结合来分析微摩尔浓度下的蛋白质复合物形成

分析微流体耦合质量光度数据以研究低亲和力蛋白质-蛋白质相互作用

首先，将微流体芯片放在质量光度计支架上并测量质量光度测量样品。完成数据采集后，打开数据分析软件。要加载文件进行分析，请单击左上角的加号图标，然后选择 mp 校准文件。软件将开始分析文件，所花费的时间可能会根据文件大小和测量次数而有所不同。要校准质量，请按位于右下角的创建质量校准按钮。将出现一个对话框，其中显示一个表格，其中包含拟合峰的对比度值。修改表格值以匹配拟合峰的已知质量值。然后点击保存。新的校准文件将显示在数据分析软件左下角的质量校准面板中。要添加示例文件，请单击左上角的加号图标，然后选择 mp 示例文件。要创建质量直方图，请导航到分析选项卡，选择直方图模式，然后选择质量绘图选项。根据需要调整垃圾桶宽度、质量限制和其他必要的参数。根据需要在图形选项卡中进一步自定义绘图。自定义后，导出图形，或将整个工作区另存为 DMP 文件。使用手动稀释进行质量光度测量得到质量直方图，其中两个最大的峰对应于未结合的 FcRn 单体和免疫球蛋白 G 抗体单体。此外，在196和251千道尔顿处清楚地观察到另外两个峰，对应于免疫球蛋白G FcRn复合物，化学计量为1：1和1：2。

analysis-microfluidics-coupled-mass-photometry-data-to-investigate

Research

JoVE Journal

Biochemistry

Analysis of the Microfluidics Coupled Mass Photometry Data to Investigate Low-Affinity Protein-Protein Interactions

246 次观看. 首先，将微流体芯片放在质量光度计支架上并测量质量光度测量样品。完成数据采集后，打开数据分析软件。要加载文件进行分析，请单击左上角的加号图标，然后选择 mp 校准文件。软件将开始分析文件，所花费的时间可能会根据文件大小和测量次数而有所不同。要校准质量，请按位于右下角的创建质量校准按钮。将出现一个对话框，其中显示一个表格，其中包含拟合...

视频: 分析微流体耦合质量光度数据以研究低亲和力蛋白质-蛋白质相互作用

Analysis of Protein Complex Formation at Micromolar Concentrations by Coupling Microfluidics with Mass Photometry

Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without labels. Mass photometry is generally suited to analyzing samples in the 100 pM-100 nM concentration range. However, in many biological systems, it is necessary to measure more concentrated samples to study low-affinity or transient interactions. Here, we demonstrate a method that effectively expands the range of sample concentrations that can be analyzed by mass photometry from nanomolar to tens of micromolar. 
In this protocol, mass photometry is combined with a novel microfluidics system to investigate the formation of protein complexes in solution in the micromolar concentration range. With the microfluidics system, users can maintain a sample at a desired higher concentration followed by dilution to the nanomolar range - several milliseconds prior to the mass photometry measurement. Due to the speed of the dilution, data is obtained before the equilibrium of the sample has shifted (i.e., dissociation of the complex). 
The technique is applied to measure interactions between an immunoglobulin G (IgG) antibody and the neonatal Fc receptor, showing the formation of high-order complexes that were not quantifiable with static mass photometry measurements. 
In conclusion, the combination of mass photometry and microfluidics makes it possible to characterize samples in the micromolar concentration range and is proficient in measuring biomolecular interactions with weaker affinities. These capabilities can be applied in a range of contexts - including the development and design of biotherapeutics - enabling thorough characterization of diverse protein-protein interactions.

This protocol combines mass photometry with a novel microfluidics system to investigate low-affinity protein-protein interactions. This approach is based on the rapid dilution of highly concentrated complexes in solution, which enables low-affinity measurements and broadens the applicability of mass photometry.

Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without ...