Recent advances in sample preparation and analysis have enabled direct profiling of protein expression in single mammalian cells and other trace samples. Several techniques to prepare and analyze ...low-input samples employ custom fluidics for nanoliter sample processing and manual sample injection onto a specialized separation column. While being effective, these highly specialized systems require significant expertise to fabricate and operate, which has greatly limited implementation in most proteomic laboratories. Here, we report a fully automated platform termed autoPOTS (automated preparation in one pot for trace samples) that uses only commercially available instrumentation for sample processing and analysis. An unmodified, low-cost commercial robotic pipetting platform was utilized for one-pot sample preparation. We used low-volume 384-well plates and periodically added water or buffer to the microwells to compensate for limited evaporation during sample incubation. Prepared samples were analyzed directly from the well plate with a commercial autosampler that was modified with a 10-port valve for compatibility with 30 μm i.d. nanoLC columns. We used autoPOTS to analyze 1–500 HeLa cells and observed only a moderate reduction in peptide coverage for 150 cells and a 24% reduction in coverage for single cells compared to our previously developed nanoPOTS platform. To evaluate clinical feasibility, we identified an average of 1095 protein groups from ∼130 sorted B or T lymphocytes. We anticipate that the straightforward implementation of autoPOTS will make it an attractive option for low-input and single-cell proteomics in many laboratories.
Fixed-time synchronization problem for a class of leader–follower multi-agent systems with second-order nonlinearity is studied in this article. A new fixed-time synchronization control algorithm is ...developed by effectively combining homogeneous system theory, Lyapunov stability theory, and fixed-time/finite-time control technology. The leader–follower multi-agent system is considered to achieve fixed-time synchronization control. Finally, numerical simulations including coordination control multiple pendulum robot systems and electric power networks are carried out to verify the control performance of the control strategy.
This study describes stationary counterflow isotachophoresis (ITP) in a poly(acrylamide‐co‐N,N′‐methylenebisacrylamide) monolithic column as a means for improving ITP processing capacity and reducing ...dispersion. The flow profile in the monolith was predicted using COMSOL's Brinkman Equation application mode, which revealed that the flow profile was mainly determined by monolith permeability. As monolith permeability decreases, the flow profile changes from a parabolic shape to a plug shape. An experimental monolithic column was prepared in a fused‐silica capillary using an ultraviolet‐initiated polymerization method. A monolithic column made from 8% (wt.) monomer was chosen for the stationary counterflow ITP experiments. Counterflow ITP in the monolithic column showed undistorted analyte zones with significantly reduced dispersion compared to the severe dispersion observed in an open capillary. Particularly, for r‐phycoerythrin focused by counterflow ITP, its zone width in the monolithic column was only one‐third that observed in an open capillary. These experiments demonstrate that stationary counterflow ITP in monoliths can be a robust and practical electrofocusing method.
Low-volume liquid handling capabilities in bioanalytical workflows can dramatically improve sample processing efficiency and reduce reagent costs, yet many commercial nanoliter liquid handlers cost ...tens of thousands of dollars or more. We have successfully adapted a low-cost and open-source commercial pipetting robot, the Opentrons OT-1, to accurately aspirate and dispense nanoliter volumes. Based on fluorescence measurements, the modified OT-1 was able to reproducibly transfer 50 nL of water with less than 3% measurement error and 5% coefficient of variation (CV). For 15 nL transfers, the volume measurements indicated less than 4% error and 4% CV. We applied this platform to the preparation of low-nanogram proteomic samples for liquid chromatography–mass spectrometry analysis, demonstrating that the modified OT-1 is an effective platform for nanoliter liquid handling. At a total materials cost of less than $6000, including the commercial liquid handler and all modifications, this system is also far less expensive than other platforms with similar capabilities, placing automated nanoliter handling within reach of a far broader scientific community.
This paper considers the constrained finite-time consensus control problem for second-order multi-agent systems with leaderless and leader–follower. By using the saturated controller design method ...and homogenous system theory, a finite-time consensus algorithm for multi-agent systems is proposed. Compared with the existing results, the proposed consensus algorithm makes the closed-loop system not only finite-time convergence but also satisfies the velocity constraints and input saturations. Simulation examples show the effectiveness of the proposed consensus algorithms.
Single-cell proteomics can provide unique insights into biological processes by resolving heterogeneity that is obscured by bulk measurements. Gains in the overall sensitivity and proteome coverage ...through improvements in sample processing and analysis increase the information content obtained from each cell, particularly for less abundant proteins. Here we report on improved single-cell proteome coverage through the combination of the previously developed nanoPOTS platform with further miniaturization of liquid chromatography (LC) separations and implementation of an ultrasensitive latest generation mass spectrometer. Following nanoPOTS sample preparation, protein digests from single cells were separated using a 20 μm i.d. in-house-packed nanoLC column. Separated peptides were ionized using an etched fused-silica emitter capable of stable operation at the ∼20 nL/min flow rate provided by the LC separation. Ultrasensitive LC–MS analysis was achieved using the Orbitrap Eclipse Tribrid mass spectrometer. An average of 362 protein groups were identified by tandem mass spectrometry (MS/MS) from single HeLa cells, and 874 protein groups were identified using the Match Between Runs feature of MaxQuant. This represents an >70% increase in label-free proteome coverage for single cells relative to previous efforts using larger bore (30 μm i.d.) LC columns coupled to a previous-generation Orbitrap Fusion Lumos mass spectrometer.
For the position synchronization control problem of multiple nonlinear manipulator systems, a bounded distributed cooperative controller is designed in this paper. Based on a fixed‐time position ...observer, a distributed finite‐time synchronization controller is designed by using the saturated control technique and homogenous system theory. Compared with the existing results, the proposed distributed cooperative control algorithm not only guarantees finite‐time convergence but also satisfies the input saturation. Finally, some numerical simulation results are presented to verify the feasibility of the theory results with six‐degree‐of‐freedom robots.
We report on the combination of nanodroplet sample preparation, ultra-low-flow nanoLC, high-field asymmetric ion mobility spectrometry (FAIMS), and the latest-generation Orbitrap Eclipse Tribrid mass ...spectrometer for greatly improved single-cell proteome profiling. FAIMS effectively filtered out singly charged ions for more effective MS analysis of multiply charged peptides, resulting in an average of 1056 protein groups identified from single HeLa cells without MS1-level feature matching. This is 2.3 times more identifications than without FAIMS and a far greater level of proteome coverage for single mammalian cells than has been previously reported for a label-free study. Differential analysis of single microdissected motor neurons and interneurons from human spinal tissue indicated a similar level of proteome coverage, and the two subpopulations of cells were readily differentiated based on single-cell label-free quantification.
The combination of nanodroplet sample preparation, ultra-low-flow nanoLC, high-field asymmetric ion mobility spectrometry (FAIMS) and latest-generation mass spectrometry instrumentation provides dramatically improved single-cell proteome profiling.
Summary
In this article, we investigate the formation control problem of multiquadrotor aircraft system subject to connectivity preservation and collision avoidance. First, based on the backstepping ...design method, a novel formation control algorithm is developed to enable multiquadrotor aircraft to move along the desired trajectory while becoming the predefined formation pattern. Meanwhile, the connectivity of the communication network is maintained and the collision among multiquadrotor is avoided. Second, for each quadrotor's desired attitude obtained by the formation controller, an improved finite‐time attitude tracking law is designed to guarantee that the desired attitude can be tracked by the real attitude in a fast finite time. Finally, a numerical example is given to demonstrate the effectiveness of the proposed algorithms.
For the position regulation problem of six degrees of freedom (6-DOF) industrial robots, a novel bounded finite-time position regulate algorithm is developed and employed to improve the dynamic ...performance of the industrial robots. First, based on the model feature, a bounded finite-time position regulate law is proposed to overcome input saturation constraints for the considered systems. Rigorous analysis guarantees the finite-time stability of the closed-loop system in the absence of disturbance. Then, to improve the disturbance rejection property of the robot systems in the presence of disturbance, an improved bounded finite-time control law based on the integral terminal sliding mode is constructed. Finally, simulations and experimental results are provided to show the effectiveness of the proposed bounded control law.
PDF
