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Related Concept Videos

Phase Diagrams02:39

Phase Diagrams

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A phase diagram combines plots of pressure versus temperature for the liquid-gas, solid-liquid, and solid-gas phase-transition equilibria of a substance. These diagrams indicate the physical states that exist under specific conditions of pressure and temperature and also provide the pressure dependence of the phase-transition temperatures (melting points, sublimation points, boiling points). Regions or areas labeled solid, liquid, and gas represent single phases, while lines or curves represent...
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Metallic Solids02:37

Metallic Solids

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Metallic solids such as crystals of copper, aluminum, and iron are formed by metal atoms. The structure of metallic crystals is often described as a uniform distribution of atomic nuclei within a “sea” of delocalized electrons. The atoms within such a metallic solid are held together by a unique force known as metallic bonding that gives rise to many useful and varied bulk properties.
All metallic solids exhibit high thermal and electrical conductivity, metallic luster, and malleability....
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Structures of Solids02:22

Structures of Solids

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Solids in which the atoms, ions, or molecules are arranged in a definite repeating pattern are known as crystalline solids. Metals and ionic compounds typically form ordered, crystalline solids. A crystalline solid has a precise melting temperature because each atom or molecule of the same type is held in place with the same forces or energy. Amorphous solids or non-crystalline solids (or, sometimes, glasses) which lack an ordered internal structure and are randomly arranged. Substances that...
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Phase Transitions02:31

Phase Transitions

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Whether solid, liquid, or gas, a substance's state depends on the order and arrangement of its particles (atoms, molecules, or ions). Particles in the solid pack closely together, generally in a pattern. The particles vibrate about their fixed positions but do not move or squeeze past their neighbors. In liquids, although the particles are closely spaced, they are randomly arranged. The position of the particles are not fixed—that is, they are free to move past their neighbors to...
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Phase Transitions: Sublimation and Deposition02:33

Phase Transitions: Sublimation and Deposition

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Some solids can transition directly into the gaseous state, bypassing the liquid state, via a process known as sublimation. At room temperature and standard pressure, a piece of dry ice (solid CO2) sublimes, appearing to gradually disappear without ever forming any liquid. Snow and ice sublimate at temperatures below the melting point of water, a slow process that may be accelerated by winds and the reduced atmospheric pressures at high altitudes. When solid iodine is warmed, the solid sublimes...
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Network Covalent Solids02:18

Network Covalent Solids

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Network covalent solids contain a three-dimensional network of covalently bonded atoms as found in the crystal structures of nonmetals like diamond, graphite, silicon, and some covalent compounds, such as silicon dioxide (sand) and silicon carbide (carborundum, the abrasive on sandpaper). Many minerals have networks of covalent bonds.
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Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue
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Universal Solid-Phase Protein Preparation (USP3) for Bottom-up and Top-down Proteomics.

Laura F Dagley1,2, Giuseppe Infusini1,2, Rune H Larsen1,2

  • 1The Walter and Eliza Hall Institute of Medical Research , Parkville , Victoria 3052 , Australia.

Journal of Proteome Research
|May 30, 2019
PubMed
Summary

A new universal, solid-phase protein preparation (USP3) method streamlines high-throughput proteomics. This robust technique enhances protein digestion and is compatible with both bottom-up and top-down mass spectrometry (MS) analysis.

Keywords:
bottom-up proteomicsintact mass spectrometrytop-down proteomicsuniversal solid-phase protein preparation (USP)

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Area of Science:

  • Proteomics
  • Analytical Chemistry
  • Biochemistry

Background:

  • Effective sample preparation is crucial for quantitative mass spectrometry (MS)-based proteomics.
  • Existing methods like filter-aided sample preparation (FASP) can be time-consuming and less efficient for high-throughput workflows.

Purpose of the Study:

  • To introduce a universal, solid-phase protein preparation (USP3) method for rapid, robust, and scalable protein sample preparation.
  • To optimize USP3 for both bottom-up and top-down MS analyses.
  • To demonstrate the scalability and efficiency of USP3 compared to existing methods.

Main Methods:

  • The universal, solid-phase protein preparation (USP3) method builds upon the single-pot solid-phase-enhanced sample preparation (SP3) technique.
  • Optimization of bead and enzyme amounts for efficient protein digestion.
  • Incorporation of acid hydrolysis for DNA/RNA removal during proteome extraction.
  • Benchmarking against filter-aided sample preparation (FASP) using HeLa protein lysate.

Main Results:

  • USP3 demonstrates scalability from low to high microgram quantities of protein.
  • The method shows efficient protein digestion and is compatible with both bottom-up and top-down MS.
  • Over 1800 proteoforms were reproducibly detected from 50 μg of HeLa protein lysate using top-down MS.
  • USP3 offers a cost-effective and robust alternative to FASP with minimal downtime.

Conclusions:

  • USP3 is a versatile and efficient sample preparation method for high-throughput proteomics.
  • The protocol enables reproducible data generation for both bottom-up and top-down MS.
  • USP3 simplifies routine analysis and reduces the time from sample collection to MS analysis.