scholarly journals High-pH reversed-phase fractionated neural retina proteome of normal growing C57BL/6 mouse

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Ying Hon Sze ◽  
Qian Zhao ◽  
Jimmy Ka Wai Cheung ◽  
King Kit Li ◽  
Dennis Yan Yin Tse ◽  
...  
Keyword(s):  
Visual Information ◽  
Protein Identification ◽  
Wild Type Mouse ◽  
Reversed Phase ◽  
Mouse Retina ◽  
Spectral Library ◽  
High Ph ◽  
Data Independent Acquisition ◽  
Proteomics Approach ◽  
Extensive Reference

AbstractThe retina is a key sensory tissue composed of multiple layers of cell populations that work coherently to process and decode visual information. Mass spectrometry-based proteomics approach has allowed high-throughput, untargeted protein identification, demonstrating the presence of these proteins in the retina and their involvement in biological signalling cascades. The comprehensive wild-type mouse retina proteome was prepared using a novel sample preparation approach, the suspension trapping (S-Trap) filter, and further fractionated with high-pH reversed phase chromatography involving a total of 28 injections. This data-dependent acquisition (DDA) approach using a Sciex TripleTOF 6600 mass spectrometer identified a total of 7,122 unique proteins (1% FDR), and generated a spectral library of 5,950 proteins in the normal C57BL/6 mouse retina. Data-independent acquisition (DIA) approach relies on a large and high-quality spectral library to analyse chromatograms, this spectral library would enable access to SWATH-MS acquisition to provide unbiased, multiplexed, and quantification of proteins in the mouse retina, acting as the most extensive reference library to investigate retinal diseases using the C57BL/6 mouse model.

scholarly journals Optimization of Spectral Library Size Improves DIA-MS Proteome Coverage

2020 ◽  
Author(s):  
Weigang Ge ◽  
Xiao Liang ◽  
Fangfei Zhang ◽  
Luang Xu ◽  
Nan Xiang ◽  
...  
Keyword(s):  
Protein Identification ◽  
A Priori ◽  
Peptide Identification ◽  
Colorectal Tumor ◽  
Spectral Library ◽  
Library Size ◽  
Test Dataset ◽  
Proteome Coverage ◽  
Data Independent Acquisition ◽  
Computational Strategy

AbstractEfficient peptide and protein identification from data-independent acquisition mass spectrometric (DIA-MS) data typically rely on an experiment-specific spectral library with a suitable size. Here, we report a computational strategy for optimizing the spectral library for a specific DIA dataset based on a comprehensive spectral library, which is accomplished by a priori analysis of the DIA dataset. This strategy achieved up to 44.7% increase in peptide identification and 38.1% increase in protein identification in the test dataset of six colorectal tumor samples compared with the comprehensive pan-human library strategy. We further applied this strategy to 389 carcinoma samples from 15 tumor datasets and observed up to 39.2% increase in peptide identification and 19.0% increase in protein identification. In summary, we present a computational strategy for spectral library size optimization to achieve deeper proteome coverage of DIA-MS data.

Imaging of retina cellular and subcellular structures using ptychographic hard X-ray tomography

2021 ◽  
Vol 134 (19) ◽  
Author(s):  
Valerie Panneels ◽  
Ana Diaz ◽  
Cornelia Imsand ◽  
Manuel Guizar-Sicairos ◽  
Elisabeth Müller ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Ion Beam ◽  
Plexiform Layer ◽  
Wild Type Mouse ◽  
Small Region ◽  
Mouse Retina ◽  
Imaging Method ◽  
Scanning Method ◽  
X Ray ◽  
Correlative Imaging

ABSTRACT Ptychographic hard X-ray computed tomography (PXCT) is a recent method allowing imaging with quantitative electron-density contrast. Here, we imaged, at cryogenic temperature and without sectioning, cellular and subcellular structures of a chemically fixed and stained wild-type mouse retina, including axons and synapses, with complete isotropic 3D information over tens of microns. Comparison with tomograms of degenerative retina from a mouse model of retinitis pigmentosa illustrates the potential of this method for analyzing disease processes like neurodegeneration at sub-200 nm resolution. As a non-destructive imaging method, PXCT is very suitable for correlative imaging. Within the outer plexiform layer containing the photoreceptor synapses, we identified somatic synapses. We used a small region inside the X-ray-imaged sample for further high-resolution focused ion beam/scanning electron microscope tomography. The subcellular structures of synapses obtained with the X-ray technique matched the electron microscopy data, demonstrating that PXCT is a powerful scanning method for tissue volumes of more than 60 cells and sensitive enough for identification of regions as small as 200 nm, which remain available for further structural and biochemical investigations.

scholarly journals Arabidopsis proteome and the mass spectral assay library

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Huoming Zhang ◽  
Pei Liu ◽  
Tiannan Guo ◽  
Huayan Zhao ◽  
Dalila Bensaddek ◽  
...  
Keyword(s):  
Quantitative Proteomics ◽  
Model Organism ◽  
Massively Parallel ◽  
Spectral Library ◽  
Mass Spectral ◽  
Novel Proteins ◽  
Data Independent Acquisition ◽  
Arabidopsis Proteome ◽  
Expression Atlas ◽  
Targeted Approach

AbstractArabidopsis is an important model organism and the first plant with its genome completely sequenced. Knowledge from studying this species has either direct or indirect applications for agriculture and human health. Quantitative proteomics by data-independent acquisition mass spectrometry (SWATH/DIA-MS) was recently developed and is considered as a high-throughput, massively parallel targeted approach for accurate proteome quantification. In this approach, a high-quality and comprehensive spectral library is a prerequisite. Here, we generated an expression atlas of 10 organs of Arabidopsis and created a library consisting of 15,514 protein groups, 187,265 unique peptide sequences, and 278,278 precursors. The identified protein groups correspond to ~56.5% of the predicted proteome. Further proteogenomics analysis identified 28 novel proteins. We applied DIA-MS using this library to quantify the effect of abscisic acid on Arabidopsis. We were able to recover 8,793 protein groups of which 1,787 were differentially expressed. MS data are available via ProteomeXchange with identifier PXD012708 and PXD012710 for data-dependent acquisition and PXD014032 for DIA analyses.

scholarly journals Reproducibility, Specificity and Accuracy of Relative Quantification Using Spectral Library-based Data-independent Acquisition

2019 ◽  
Vol 19 (1) ◽  
pp. 181-197 ◽  
Author(s):  
Katalin Barkovits ◽  
Sandra Pacharra ◽  
Kathy Pfeiffer ◽  
Simone Steinbach ◽  
Martin Eisenacher ◽  
...  
Keyword(s):  
Relative Quantification ◽  
Spectral Library ◽  
Data Independent Acquisition

scholarly journals Detection of Functional Overreaching in Endurance Athletes Using Proteomics

Proteomes ◽  
2018 ◽  
Vol 6 (3) ◽  
pp. 33 ◽  
Author(s):  
David Nieman ◽  
Arnoud Groen ◽  
Artyom Pugachev ◽  
Gianmarco Vacca
Keyword(s):  
Estimating Equation ◽  
Innate Immune ◽  
Generalized Estimating Equation ◽  
Blood Proteins ◽  
Post Exercise ◽  
Data Independent Acquisition ◽  
Immune System Activation ◽  
Fasted State ◽  
Proteomics Approach ◽  
System Activation

No reliable biomarkers exist to identify athletes in various training states including functional overreaching (FOR), non-functional overreaching (NFOR), and overtraining syndrome (OTS). Participants (N = 10, age 38.3 ± 3.4 years) served as their own controls and in random, counterbalanced order either ran/cycled 2.5 h (70.0 ± 3.7% VO2max) three days in a row (FOR) or sat in the lab (rest) (separated by three weeks; 7:00–9:30 am, overnight fasted state). Participants provided fingerprick samples for dried blood spot samples (DBS) pre- and post-exercise/rest, and then during two recovery days. DBS proteins were measured with nanoLC-MS in data-independent acquisition (DIA) mode, and 593 proteins were identified and quantified. Proteins were considered for the FOR cluster if they were elevated during one of the two recovery days but not more than one of the exercise days (compared to rest). The generalized estimating equation (GEE) was used to identify proteins linked to FOR. A total of 13 proteins was linked to FOR and most were associated with the acute phase response and innate immune system activation. This study used a system-wide proteomics approach to define a targeted panel of blood proteins related to FOR that could form the basis of future NFOR- and OTS-based studies.

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