Part II: Identification of Proteins with MALDI-TOF

Target Sample Deposition

Classic Dry Liquid Droplet Method

Step I: Preparation of semi-saturated cyano-4-hydroxycinnamic acid (approximately 5 mg / ml): Dissolve α-cyano-4-hydroxybenzoic acid in 300 μl of 1: 1 acetonitrile / TFA solution (ultrasonication for 10 min). Centrifuge the liquid for several seconds to obtain the supernatant. Add 200 μl of the supernatant to a microcentrifuge tube, and add the same volume (200 μl) of a 1: 1 acetonitrile / TFA solution to prepare a semi-saturated α-cyano-4-hydroxycinnamic acid.

Step II: Take 0.8 μl of digestion solution and 0.8 μl of matrix into a microcentrifuge tube and mix them quickly (avoid crystallization at the tip of the pipette). Take 0.8 μl of the mixed solution onto the MALDI base. The remaining 0.8 μl of the mixed solution can be dropped on another position of the MALDI base, and the tip of the pipette should not touch the standard.

Step III: Let the mixture dry naturally and crystallize.

Method of Drying Liquid Droplet on Hydrophobe Base

Step I: Prepare α-cyano-4-hydroxycinnamic acid: Weigh 10 mg of α-cyano-4-hydroxycinnamic acid and dissolve it in 1 ml of a 1: 1 acetonitrile / TFA solution (ultrasonication for 10 min). Take 56 μl of the above solution into a new microcentrifuge tube, and add 994 μl of a 1: 1 acetonitrile / TFA solution.

Step II: Quickly mix 0.8 μl of the gel-digested supernatant and 0.8 μl of the above solution.Take another 0.8 μl of the mixed solution onto the MALDI base, and the remaining 0.8 μl of the mixed solution can be dropped on another position of the MALDI standard.

Step III: Allow the mixture to dry naturally and crystallize.

Step IV: Add 4 μl of TFA solution to the crystals. After 30 seconds of contact, suck up the liquid with a pipette (the tip of the pipette should not touch the crystal).

Step V: Add 0.8 μl of ethanol / acetone / TFA solution and crystallize again.

Step VI: Allow the mixture to dry naturally and crystallize.

Spectrum Acquisition

Step I: Insert the base into the mass spectrometer and stabilize in vacuum (less than 10-6 Torr).

Step II: Turn on the pressure (It is best to wait for 20 minutes to make the voltage and temperature constant (Joule effect)).

Step III: Adjust the laser power (attenuation) and target position so that a good signal-to-noise ratio and a single isotope resolution can be obtained at 700 ~ 4000 Th.

Step IV: Lase 10 times, but the spectra gets is not so valuable because they are usually images of small mass molecules (matrix and / or salt).

Step V: Lase 80 ~ 200 times, superpose the spectrum acquired.

Step VI: Obtain ions using hydrolyzed trypsin at 842.5099 Th and 2211.1046 Th, and perform internal correction by ms.

Step VII: Save the spectrum.

Spetrum Analysis

A single isotope mass can be assigned automatically, but a careful check to these resolutions is recommend.

If the map is not smoothed, all primary single isotope peptide peaks can be resolved, except for peaks generated by known trypsin.

The evaluation criteria of the candidate protein are as follows:

The score is better than the critical threshold.
There is a large difference between the optimal protein and the unrelated protein.
The protein of the species (or similar species with high homology) is the first class of optimal protein.
The molecular weight and isoelectric point of the candidate proteins are compatible with two-dimensional gel data.
Maximization of three matched peptides by one cleaved peptide.
Minimization of 5 matching peptides.

MALDI-TOF/TOF Strategy

The proteins identified by PMF are only preliminary candidates, and the score of the identified protein indicates the degree of confidence. Therefore, secondary sequencing should be further performed on the ion peaks obtained from PMF to improve and confirm the reliability of protein identification.

Before the recent invention of the TOF / TOF tandem mass spectrometer, the success rate of sequencing peptides on a MALDI mass spectrometer was poor. On the TOF / TOF analyzer, the first-level TOF can separate the peptide ions we need, and the other peptide ions are removed directly. After screening by the first-level TOF bombardment, the remaining peptides are specific amino acid sequence peptides. The secondary TOF then separates and measures the molecular mass of these specific amino acid sequence fragments.

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