scholarly journals Improved LC-MS chromatographic alignment increases the accuracy of label-free quantitative proteomics: Comparison of spectral counting versus ion intensity-based proteomic quantification strategies

2017 ◽  
Author(s):  
Daniel H.J. Ng ◽  
Jonathan D. Humphries ◽  
Julian N. Selley ◽  
Stacey Warwood ◽  
David Knight ◽  
...  
Keyword(s):  
Cell Biology ◽  
Low Cost ◽  
Quantitative Description ◽  
Protein Quantification ◽  
Global Changes ◽  
Label Free ◽  
Spectral Counting ◽  
Differential Abundance ◽  
Mass Spectrometers ◽  
Wide Range

AbstractThe ability to provide an unbiased qualitative and quantitative description of the global changes to proteins in a cell or an organism would permit the systems-wide study of complex biological systems. Label-free quantitative shotgun proteomic strategies (including LC-MS ion intensity quantification and spectral counting) are attractive because of their relatively low cost, ease of implementation, and the lack of multiplexing restrictions when comparing multiple samples. Owing to improvements in the resolution and sensitivity of mass spectrometers, and the availability of analytical software packages, protein quantification by LC-MS ion intensity has increased in popularity. Here, we have addressed the importance of chromatographic alignment on protein quantification, and then assessed how spectral counting compares to ion intensity-based proteomic quantification. Using a spiked-in protein strategy, we analysed two situations that commonly arise in the application of proteomics to cell biology: (i) samples with a small number of proteins of differential abundance in a larger non-changing background, and (ii) samples with a larger number of proteins of differential abundance. To perform these assessments on biologically relevant samples, we used isolated integrin adhesion complexes (IACs). Technical replicate analysis of isolated IACs resulted in a range of alignment scores using the Progenesis QI software package and demonstrated that higher LC-MS chromatographic alignment scores increased the precision of protein quantification. Furthermore, implementation of a simple sample batch-running strategy enabled good chromatographic alignment for hundreds of samples over multiple batches. Finally, we applied the sample batch-running strategy and compared quantification by LC-MS ion intensity to spectral counting and found that quantification by LC-MS ion intensity was more accurate and precise. In summary, these results demonstrate that chromatographic alignment is important for precise and accurate protein quantification based on LC-MS ion intensity and accordingly we present a simple sample re-ordering strategy to facilitate improved alignment. These findings are not only relevant to label-free quantification using Progenesis QI but may be useful to the wide range of MS-based quantification strategies that rely on chromatographic alignment.

A simple device to apply equibiaxial strain to cells cultured on flexible membranes

2008 ◽  
Vol 294 (1) ◽  
pp. H532-H540 ◽  
Author(s):  
Obaida R. Rana ◽  
Carsten Zobel ◽  
Esra Saygili ◽  
Klara Brixius ◽  
Felix Gramley ◽  
...  
Keyword(s):  
Cell Biology ◽  
Low Cost ◽  
Normal Growth ◽  
Neonatal Rat ◽  
Simple Device ◽  
Static Stretch ◽  
Voltage Gated ◽  
Wide Range ◽  
Dose Dependent ◽  
Cells Cultured

The biomechanical environment to which cells are exposed is important to their normal growth, development, interaction, and function. Accordingly, there has been much interest in studying the role of biomechanical forces in cell biology and pathophysiology. This has led to the introduction and even commercialization of many experimental devices. Many of the early devices were limited by the heterogeneity of deformation of cells cultivated in different locations of the culture plate membranes and were also attached with complicated technical/electronic efforts resulting in a restriction of the reproducibility of these devices. The objective of this study was to design and build a simple device to allow the application of dose-dependent homogeneous equibiaxial static stretch to cells cultured on flexible silicone membranes to investigate biological and biomedical questions. In addition, cultured neonatal rat atrial cardiomyocytes were stretched with the proposed device with different strain gradients. For the first time with this study we could demonstrate that stretch up to 21% caused dose-dependent changes in biological markers such as the calcineurin activity, modulatory calcineurin-interacting protein-1, voltage-gated potassium channel isoform 4.2, and voltage-gated K+ channel-interacting proteins-2 gene expression and transient outward potassium current densities but not the protein-to-DNA ratio and atrial natriuretic peptide mRNA. With both markers mentioned last, dose-dependent stretch alterations could only be achieved with stretch up to 13%. The simple and low-cost device presented here might be applied to a wide range of experimental settings in different fields of research.

scholarly journals Integrating identification and quantification uncertainty for differential protein abundance analysis with Triqler

2020 ◽  
Author(s):  
Matthew The ◽  
Lukas Käll
Keyword(s):  
Missing Values ◽  
Shotgun Proteomics ◽  
Protein Quantification ◽  
Protein Abundance ◽  
Posterior Distributions ◽  
Label Free ◽  
Differential Abundance ◽  
Complicated Process ◽  
Python Package ◽  
Different Parts

AbstractProtein quantification for shotgun proteomics is a complicated process where errors can be introduced in each of the steps. Triqler is a Python package that estimates and integrates errors of the different parts of the label-free protein quantification pipeline into a single Bayesian model. Specifically, it weighs the quantitative values by the confidence we have in the correctness of the corresponding PSM. Furthermore, it treats missing values in a way that reflects their uncertainty relative to observed values. Finally, it combines these error estimates in a single differential abundance FDR that not only reflects the errors and uncertainties in quantification but also in identification. In this tutorial, we show how to (1) generate input data for Triqler from quantification packages such as MaxQuant and Quandenser, (2) run Triqler and what the different options are, (3) interpret the results, (4) investigate the posterior distributions of a protein of interest in detail and (5) verify that the hyperparameter estimations are sensible.

scholarly journals Colorimetric Sensing with Gold Nanoparticles on Electrowetting-Based Digital Microfluidics

Micromachines ◽  
2021 ◽  
Vol 12 (11) ◽  
pp. 1423
Author(s):  
Zhen Gu ◽  
Jing-Jing Luo ◽  
Le-Wei Ding ◽  
Bing-Yong Yan ◽  
Jia-Le Zhou ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Low Cost ◽  
Digital Microfluidics ◽  
High Sensitivity ◽  
Bulk Solution ◽  
Label Free ◽  
Colorimetric Sensing ◽  
Wide Range ◽  
The Stability ◽  
Digital Microfluidic

Digital microfluidic (DMF) has been a unique tool for manipulating micro-droplets with high flexibility and accuracy. To extend the application of DMF for automatic and in-site detection, it is promising to introduce colorimetric sensing based on gold nanoparticles (AuNPs), which have advantages including high sensitivity, label-free, biocompatibility, and easy surface modification. However, there is still a lack of studies for investigating the movement and stability of AuNPs for in-site detection on the electrowetting-based digital microfluidics. Herein, to demonstrate the ability of DMF for colorimetric sensing with AuNPs, we investigated the electrowetting property of the AuNPs droplets on the hydrophobic interface of the DMF chip and examined the stability of the AuNPs on DMF as well as the influence of evaporation to the colorimetric sensing. As a result, we found that the electrowetting of AuNPs fits to a modified Young–Lippmann equation, which suggests that a higher voltage is required to actuate AuNPs droplets compared with actuating water droplets. Moreover, the stability of AuNPs was maintained during the processing of electrowetting. We also proved that the evaporation of droplets has a limited influence on the detections that last several minutes. Finally, a model experiment for the detection of Hg2+ was carried out with similar results to the detections in bulk solution. The proposed method can be further extended to a wide range of AuNPs-based detection for label-free, automatic, and low-cost detection of small molecules, biomarkers, and metal ions.

scholarly journals A hierarchical 3D-motion learning framework for animal spontaneous behavior mapping

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kang Huang ◽  
Yaning Han ◽  
Ke Chen ◽  
Hongli Pan ◽  
Gaoyang Zhao ◽  
...  
Keyword(s):  
Low Cost ◽  
Disease Model ◽  
Quantitative Description ◽  
Feature Space ◽  
Transgenic Animal ◽  
Neural System ◽  
Animal Disease ◽  
Animal Disease Model ◽  
Wide Range ◽  
Spontaneous Behavior

AbstractAnimal behavior usually has a hierarchical structure and dynamics. Therefore, to understand how the neural system coordinates with behaviors, neuroscientists need a quantitative description of the hierarchical dynamics of different behaviors. However, the recent end-to-end machine-learning-based methods for behavior analysis mostly focus on recognizing behavioral identities on a static timescale or based on limited observations. These approaches usually lose rich dynamic information on cross-scale behaviors. Here, inspired by the natural structure of animal behaviors, we address this challenge by proposing a parallel and multi-layered framework to learn the hierarchical dynamics and generate an objective metric to map the behavior into the feature space. In addition, we characterize the animal 3D kinematics with our low-cost and efficient multi-view 3D animal motion-capture system. Finally, we demonstrate that this framework can monitor spontaneous behavior and automatically identify the behavioral phenotypes of the transgenic animal disease model. The extensive experiment results suggest that our framework has a wide range of applications, including animal disease model phenotyping and the relationships modeling between the neural circuits and behavior.

scholarly journals Label-Free Assays on the BIND System

2004 ◽  
Vol 9 (6) ◽  
pp. 481-490 ◽  
Author(s):  
Brian T. Cunningham ◽  
Peter Li ◽  
Stephen Schulz ◽  
Bo Lin ◽  
Cheryl Baird ◽  
...  
Keyword(s):  
Fluorescent Dyes ◽  
Dynamic Range ◽  
Low Cost ◽  
High Sensitivity ◽  
Receptor Expression ◽  
Label Free ◽  
Protein Protein Interaction ◽  
Small Molecule Screening ◽  
Guided Mode ◽  
Wide Range

Screening of biochemical interactions becomes simpler, less expensive, and more accurate when labels, such as fluorescent dyes, radioactive markers, and colorimetric reactions, are not required to quantify detected material. SRU Biosystems has developed a biosensor technology that is manufactured on continuous sheets of plastic film and incorporated into standard microplates and microarray slides to enable label-free assays to be performed with high throughput, high sensitivity, and low cost per assay. The biosensor incorporates a narrow band guided-mode resonance reflectance filter, in which the reflected color is modulated by the attachment/detachment of biochemical material to the surface. The technology offers 4 orders of linear dynamic range and uniformity within a plate, with a coefficient of variation of 2.5%. Using conventional biochemical immobilization surface chemistries, a wide range of assay applications are enabled. Small molecule screening, cell proliferation/cytotoxicity, enzyme activity screening, protein-protein interaction, and cell membrane receptor expression are among the applications demonstrated.

scholarly journals Cellular uptake and dynamics of unlabeled freestanding silicon nanowires

2016 ◽  
Vol 2 (12) ◽  
pp. e1601039 ◽  
Author(s):  
John F. Zimmerman ◽  
Ramya Parameswaran ◽  
Graeme Murray ◽  
Yucai Wang ◽  
Michael Burke ◽  
...  
Keyword(s):  
Silicon Nanowires ◽  
Cell Biology ◽  
Valuable Insight ◽  
Dependent Manner ◽  
Label Free ◽  
Semiconductor Nanowire ◽  
Wide Range ◽  
Intracellular Sensing ◽  
Device Integration ◽  
Multiple Cell

The ability to seamlessly merge electronic devices with biological systems at the cellular length scale is an exciting prospect for exploring new fundamental cell biology and in designing next-generation therapeutic devices. Semiconductor nanowires are well suited for achieving this goal because of their intrinsic size and wide range of possible configurations. However, current studies have focused primarily on delivering substrate-bound nanowire devices through mechanical abrasion or electroporation, with these bulkier substrates negating many of the inherent benefits of using nanoscale materials. To improve on this, an important next step is learning how to distribute these devices in a drug-like fashion, where cells can naturally uptake and incorporate these electronic components, allowing for truly noninvasive device integration. We show that silicon nanowires (SiNWs) can potentially be used as such a system, demonstrating that label-free SiNWs can be internalized in multiple cell lines (96% uptake rate), undergoing an active “burst-like” transport process. Our results show that, rather than through exogenous manipulation, SiNWs are internalized primarily through an endogenous phagocytosis pathway, allowing cellular integration of these materials. To study this behavior, we have developed a robust set of methodologies for quantitatively examining high–aspect ratio nanowire-cell interactions in a time-dependent manner on both single-cell and ensemble levels. This approach represents one of the first dynamic studies of semiconductor nanowire internalization and offers valuable insight into designing devices for biomolecule delivery, intracellular sensing, and photoresponsive therapies.

scholarly journals Electrospun Nanofibers for Label-Free Sensor Applications

Sensors ◽  
2019 ◽  
Vol 19 (16) ◽  
pp. 3587 ◽  
Author(s):  
Nahal Aliheidari ◽  
Nojan Aliahmad ◽  
Mangilal Agarwal ◽  
Hamid Dalir
Keyword(s):  
Low Cost ◽  
High Surface ◽  
Volume Ratio ◽  
Electrospun Nanofibers ◽  
Label Free ◽  
Sensor Applications ◽  
Wide Range ◽  
Sensor Properties ◽  
Fundamental Research ◽  
Future Work

Electrospinning is a simple, low-cost and versatile method for fabricating submicron and nano size fibers. Due to their large surface area, high aspect ratio and porous structure, electrospun nanofibers can be employed in wide range of applications. Biomedical, environmental, protective clothing and sensors are just few. The latter has attracted a great deal of attention, because for biosensor application, nanofibers have several advantages over traditional sensors, including a high surface-to-volume ratio and ease of functionalization. This review provides a short overview of several electrospun nanofibers applications, with an emphasis on biosensor applications. With respect to this area, focus is placed on label-free sensors, pertaining to both recent advances and fundamental research. Here, label-free sensor properties of sensitivity, selectivity, and detection are critically evaluated. Current challenges in this area and prospective future work is also discussed.

scholarly journals Hartman interferometer: versatile integrated optic sensor for label-free, real-time quantification of nucleic acids, proteins, and pathogens

1997 ◽  
Vol 43 (9) ◽  
pp. 1757-1763 ◽  
Author(s):  
Bernard H Schneider ◽  
John G Edwards ◽  
Nile F Hartman
Keyword(s):  
Real Time ◽  
Optical Sensors ◽  
Evanescent Wave ◽  
Low Cost ◽  
Refractive Index Change ◽  
Polarized Light ◽  
Gene Probe ◽  
Label Free ◽  
Optic Sensor ◽  
Wide Range

Abstract The Hartman interferometer, a proprietary integrated optic sensor, provides a basis for a broad range of biomedical diagnostics, including antibody-based and gene probe-based assays. As with other evanescent-wave optical sensors, the interferometer measures the refractive index change resulting from biomolecular binding on a waveguide surface. The exciting promise of evanescent-wave sensors lies, in general, in their potential to be used as label-free, real-time transducers that can operate in a true mix-and-read fashion and provide fast, quantitative results. One of the major issues facing their development, however, is creating a simple, low-cost configuration for multianalyte testing. The Hartman interferometer addresses this challenge by relying on linearly polarized light and a planar waveguide format, thereby avoiding the problems associatedwith circular polarization and channel waveguides. We report preliminary experiments that demonstrate the applicability of this sensor configuration to detection of a wide range of protein, nucleic acid, and pathogen analytes.

scholarly journals Developing Nanodisc-ID for label-free characterizations of membrane proteins

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Huan Bao
Keyword(s):  
Membrane Proteins ◽  
Protein Interactions ◽  
Gel Electrophoresis ◽  
Low Cost ◽  
Label Free ◽  
Protein Protein Interactions ◽  
Peripheral Membrane Proteins ◽  
Wide Range ◽  
Neural Transmission

AbstractMembrane proteins (MPs) influence all aspects of life, such as tumorigenesis, immune response, and neural transmission. However, characterization of MPs is challenging, as it often needs highly specialized techniques inaccessible to many labs. We herein introduce nanodisc-ID that enables quantitative analysis of membrane proteins using a gel electrophoresis readout. By leveraging the power of nanodiscs and proximity labeling, nanodisc-ID serves both as scaffolds for encasing biochemical reactions and as sensitive reagents for detecting membrane protein-lipid and protein-protein interactions. We demonstrate this label-free and low-cost tool by characterizing a wide range of integral and peripheral membrane proteins from prokaryotes and eukaryotes.

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