Experiments for precise molecular weight determination of proteins
Experiments for precise molecular weight determination of proteins
Summary
The exact molecular weight of a protein is the sum of all the atomic weights contained in the molecule. Currently, there is one main method used to determine the exact molecular weight of a protein: mass spectrometry to determine the exact molecular weight of a protein.
Principle
The basic principle of mass spectrometry for determining the precise molecular weight of proteins is to ionize proteins and matrix molecules in an ion source, transfer protons from the matrix to the proteins, generate positively charged ions with different charge ratios, and form ion beams by the action of an accelerating electric field, which enter a mass analyzer. In the electric and magnetic fields of the mass analyzer, the ion beam undergoes opposite velocity dispersion and focusing to obtain the mass spectra, thus determining the mass of the protein molecules.
Operation method
Precise molecular weight determination of proteins by mass spectrometry
Materials and Instruments
Equipment: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry
Reagents:
① ultrapure water
② Matrix buffer containing 50% acetonitrile, 0.1% trifluoroacetic acid
③ Saturated matrix solution (saturated α-cyano-4-hydroxycinnamic acid)
Move
The basic process of determining the exact molecular weight of a protein by mass spectrometry can be divided into the following steps. In this method, matrix-assisted laser desorption ionization time-of-flight mass spectrometry is used as an example to introduce the whole process of the mass spectrometry technique.
A. The purified protein is dialyzed in ultrapure water to remove the salt content, and then vacuum freeze-dried into dry protein powder.
B. The protein was analyzed with ultrapure water or matrix buffer. B. Dissolve the protein powder in ultrapure water or matrix buffer containing 50% acetonitrile and 0.1% trifluoroacetic acid. B. Dissolve the dried protein powder in ultrapure water or matrix buffer containing 50% acetonitrile, 0.1% trifluoroacetic acid and dilute to 0.1-10pmol/μl.
C. Select a suitable calibrated protein standard molecule mixture with a molecular weight range that covers the molecular mass of the sample to be tested.
D. Combine the protein sample solution with a saturated matrix solution (saturated α-hydroxybenzene). (saturated with α-cyano-4-hydroxycinnamic acid). D. Mix the protein sample solution with the saturated matrix solution (saturated α-cyano-4-hydroxycinnamic acid) in a 1:1 ratio.
E. Apply 1 μl of the above mixture to the sample target and air dry.
F. Place the sample target containing the protein standard and the sample protein into the MALDI-TOF-MS mass spectrometer and select the appropriate test method to obtain the mass spectra and protein molecular weight. Compare the difference between the measured exact molecular weight of the target protein molecule and the relative molecular weight deduced from the amino acid sequence of the protein.
Caveat
1. The optimal concentration for different protein molecules may vary. For common protein samples, the concentration should be controlled at 0.1~10mmol/L; for some glycoproteins, the detection effect is better when the concentration is more than 10mmol/L; for proteins with molecular weight more than 50kDa, the concentration is more than 100mmol/L to get better detection results.
2. Purified proteins must be desalted to remove components that interfere with crystallization and ionization. Some contaminants such as EDTA, glycerol, sodium azide, and detergents can interfere with the sensitivity of the assay.
3. choose the appropriate matrix according to the molecular weight of the protein, commonly used matrix is erucic acid, suitable for proteins greater than 10kDa or multi-skin;α-Cyano-4-hydroxycinnamic acidsuitable for proteins less than2,5-dihydroxybenzoic acid is suitable for the analysis of peptides and glycoproteins larger than 3kDa.
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