CSF Sample Preparation for Data-Independent Acquisition

2019 ◽  
pp. 61-67
Author(s):  
Katalin Barkovits ◽  
Lars Tönges ◽  
Katrin Marcus
Keyword(s):  
Sample Preparation ◽  
Data Independent Acquisition

scholarly journals Matrix-matched calibration curves for assessing analytical figures of merit in quantitative proteomics

2019 ◽  
Author(s):  
Lindsay K Pino ◽  
Han-Yin Yang ◽  
William Stafford Noble ◽  
Brian C Searle ◽  
Andrew N Hoofnagle ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Quantitative Proteomics ◽  
Protein Abundance ◽  
Complex Samples ◽  
Figures Of Merit ◽  
Data Independent Acquisition ◽  
Proteomics Experiment ◽  
Series Of Experiments ◽  
Analytical Figures Of Merit

AbstractMass spectrometry is a powerful tool for quantifying protein abundance in complex samples. Advances in sample preparation and the development of data independent acquisition (DIA) mass spectrometry approaches have increased the number of peptides and proteins measured per sample. Here we present a series of experiments demonstrating how to assess whether a peptide measurement is quantitative by mass spectrometry. Our results demonstrate that increasing the number of detected peptides in a proteomics experiment does not necessarily result in increased numbers of peptides that can be measured quantitatively.

scholarly journals Time-resolved in vivo ubiquitinome profiling by DIA-MS reveals USP7 targets on a proteome-wide scale

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Martin Steger ◽  
Vadim Demichev ◽  
Mattias Backman ◽  
Uli Ohmayer ◽  
Phillip Ihmor ◽  
...  
Keyword(s):  
Neural Network ◽  
Mass Spectrometry ◽  
Sample Preparation ◽  
Mode Of Action ◽  
System Level ◽  
High Temporal Resolution ◽  
Time Resolved ◽  
Data Independent Acquisition ◽  
Wide Scale

AbstractMass spectrometry (MS)-based ubiquitinomics provides system-level understanding of ubiquitin signaling. Here we present a scalable workflow for deep and precise in vivo ubiquitinome profiling, coupling an improved sample preparation protocol with data-independent acquisition (DIA)-MS and neural network-based data processing specifically optimized for ubiquitinomics. Compared to data-dependent acquisition (DDA), our method more than triples identification numbers to 70,000 ubiquitinated peptides in single MS runs, while significantly improving robustness and quantification precision. Upon inhibition of the oncology target USP7, we simultaneously record ubiquitination and consequent changes in abundance of more than 8,000 proteins at high temporal resolution. While ubiquitination of hundreds of proteins increases within minutes of USP7 inhibition, we find that only a small fraction of those are ever degraded, thereby dissecting the scope of USP7 action. Our method enables rapid mode-of-action profiling of candidate drugs targeting DUBs or ubiquitin ligases at high precision and throughput.

scholarly journals Mass spectrometry-based proteomic analysis of NSCLC tumor and biopsy samples

2021 ◽  
Author(s):  
Janne Lehtiö ◽  
Taner Arslan ◽  
Ioannis Siavelis ◽  
Yanbo Pan ◽  
Fabio Socciarelli ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Molecular Subtypes ◽  
Solid Phase ◽  
Median Number ◽  
Label Free ◽  
Biopsy Material ◽  
Entire Cohort ◽  
Data Independent Acquisition ◽  
Depth Analysis

Abstract The associated publication reports proteogenomic analysis of non-small cell lung cancer, where we identified molecular subtypes with distinct immune evasion mechanisms and therapeutic targets, and validated our classification method in separate clinical cohorts. This protocol describes the sample preparation and mass spectrometry (MS)-based in-depth and rapid proteomic analyses of tumor and biopsy samples. We deployed single-pot solid-phase-enhanced sample preparation (SP3). For the in-depth analysis, we used TMT labeling, followed by high-resolution isoelectric focusing (HiRIEF) prefractionation and LC-MS with data-dependent acquisition (DDA). The reported protocol achieved analytical depth of close to 14,000 quantified proteins and almost 10,000 across the entire cohort of 141 samples. The rapid analysis was label-free, based on LC-MS with data-independent acquisition (DIA). The median number of identified proteins was 3,967 and 3,552 in two independent cohorts of tumor samples (n = 141 and 208, respectively), and 2,494 in another cohort of biopsy material (n = 84).

Pollutant Identification by Selected Area Electron Diffraction

1974 ◽  
Vol 32 ◽  
pp. 532-533
Author(s):  
R. E. Ferrell ◽  
G. G. Paulson ◽  
C. W. Walker
Keyword(s):  
Thermal Treatment ◽  
Electron Diffraction ◽  
Sample Preparation ◽  
Crystal Structures ◽  
Water Samples ◽  
Environmental Samples ◽  
Short Review ◽  
Selected Area Electron Diffraction ◽  
Multiple Component

Selected area electron diffraction (SAD) has been used successfully to determine crystal structures, identify traces of minerals in rocks, and characterize the phases formed during thermal treatment of micron-sized particles. There is an increased interest in the method because it has the potential capability of identifying micron-sized pollutants in air and water samples. This paper is a short review of the theory behind SAD and a discussion of the sample preparation employed for the analysis of multiple component environmental samples.

Preparation of Thin Cadmium Telluride Samples for Electron Microscope Studies

1974 ◽  
Vol 32 ◽  
pp. 548-549
Author(s):  
T. J. Magee ◽  
J. Peng ◽  
J. Bean
Keyword(s):  
Electron Microscope ◽  
Sulfuric Acid ◽  
Solid State ◽  
Sample Preparation ◽  
Cadmium Telluride ◽  
Potassium Dichromate ◽  
Optical Components ◽  
The Past ◽  
Solid State Devices

Cadmium telluride has become increasingly important in a number of technological applications, particularly in the area of laser-optical components and solid state devices, Microstructural characterizations of the material have in the past been somewhat limited because of the lack of suitable sample preparation and thinning techniques. Utilizing a modified jet thinning apparatus and a potassium dichromate-sulfuric acid thinning solution, a procedure has now been developed for obtaining thin contamination-free samples for TEM examination.

A Freeze Shadow Technique for the Preparation of Soft Paint Latexes for Electron Microscopy

1977 ◽  
Vol 35 ◽  
pp. 314-315
Author(s):  
Earl R. Walter ◽  
Glen H. Bryant
Keyword(s):  
Sample Preparation ◽  
High Vacuum ◽  
Vacuum System ◽  
Latex Particles ◽  
New Methods ◽  
Diffusion Pump ◽  
Film Forming ◽  
Routine Basis ◽  
Distribution Studies ◽  
Preparation Techniques

With the development of soft, film forming latexes for use in paints and other coatings applications, it became desirable to develop new methods of sample preparation for latex particle size distribution studies with the electron microscope. Conventional latex sample preparation techniques were inadequate due to the pronounced tendency of these new soft latex particles to distort, flatten and fuse on the substrate when they dried. In order to avoid these complications and obtain electron micrographs of undistorted latex particles of soft resins, a freeze-dry, cold shadowing technique was developed. The method has now been used in our laboratory on a routine basis for several years.The cold shadowing is done in a specially constructed vacuum system, having a conventional mechanical fore pump and oil diffusion pump supplying vacuum. The system incorporates bellows type high vacuum valves to permit a prepump cycle and opening of the shadowing chamber without shutting down the oil diffusion pump. A baffeled sorption trap isolates the shadowing chamber from the pumps.

SEM evaluation of the TEM cold-stage double-film specimen

1984 ◽  
Vol 42 ◽  
pp. 272-273
Author(s):  
Jayesh Bellare
Keyword(s):  
Spatial Distribution ◽  
Sample Preparation ◽  
Sample Thickness ◽  
Specimen Preparation ◽  
Preparation Technique ◽  
Liquid Sample ◽  
Thin Specimen ◽  
Sample Preparation Technique ◽  
Cold Stage ◽  
Film Specimen

Seeing is believing, but only after the sample preparation technique has received a systematic study and a full record is made of the treatment the sample gets.For microstructured liquids and suspensions, fast-freeze thermal fixation and cold-stage microscopy is perhaps the least artifact-laden technique. In the double-film specimen preparation technique, a layer of liquid sample is trapped between 100- and 400-mesh polymer (polyimide, PI) coated grids. Blotting against filter paper drains excess liquid and provides a thin specimen, which is fast-frozen by plunging into liquid nitrogen. This frozen sandwich (Fig. 1) is mounted in a cooling holder and viewed in TEM.Though extremely promising for visualization of liquid microstructures, this double-film technique suffers from a) ireproducibility and nonuniformity of sample thickness, b) low yield of imageable grid squares and c) nonuniform spatial distribution of particulates, which results in fewer being imaged.

Advanced TEM sample preparation techniques for submicron Si devices

1995 ◽  
Vol 53 ◽  
pp. 516-517 ◽  
Author(s):  
P. B. Basham ◽  
H. L. Tsai
Keyword(s):  
Sample Preparation ◽  
Process Development ◽  
Light Transmission ◽  
Specific Area ◽  
Turnaround Time ◽  
Small Hole ◽  
Area Of Interest ◽  
Transmission Electron ◽  
Polished Side ◽  
Preparation Techniques

The use of transmission electron microscopy (TEM) to support process development of advanced microelectronic devices is often challenged by a large amount of samples submitted from wafer fabrication areas and specific-spot analysis. Improving the TEM sample preparation techniques for a fast turnaround time is critical in order to provide a timely support for customers and improve the utilization of TEM. For the specific-area sample preparation, a technique which can be easily prepared with the least amount of effort is preferred. For these reasons, we have developed several techniques which have greatly facilitated the TEM sample preparation.For specific-area analysis, the use of a copper grid with a small hole is found to be very useful. With this small-hole grid technique, TEM sample preparation can be proceeded by well-established conventional methods. The sample is first polished to the area of interest, which is then carefully positioned inside the hole. This polished side is placed against the grid by epoxy Fig. 1 is an optical image of a TEM cross-section after dimpling to light transmission.

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