Experimental material: sequencing grade porcine trypsin
Reagents: ammonium bicarbonate ammonium bicarbonate buffer acetonitrile, ammonium bicarbonate buffer digestion buffer
Instruments: vacuum centrifuge oven
Experimental procedures: Many gel staining methods are applicable to subsequent MALDI-TOF peptide mass fingerprint (PMF) analysis.
The gel digestion method was adapted from Jensen.
In gel treatment and digestion, samples must be carefully processed in order to minimize exogenous keratin contamination (eg hair, skin, wool clothes, airflow, ventilation cooling, etc.).
1. Dig protein spots from two-dimensional polyacrylamide gel
(1) Use a blade to cut 5 mm from the tip of a 1 ml pipette to make a punch (1 to 2 mm incision diameter).
(2)Dig the gel points and transfer them to the centrifuge tube.
*2. Wash gel fragments *
(1)Wash with 100 μl of ammonium bicarbonate buffer (oscillation for 15 min) and discard the supernatant.
(2)Wash twice with 100 μl acetonitrile/ammonium bicarbonate buffer (oscillation for 15 min) and discard the supernatant.
(3)Shrink the gel fragments with 100 μl of HPLC grade acetonitrile (oscillation for 15 min) and discard the supernatant.
(4)Dry the gel fragments by vacuum drying on a centrifugal evaporator.
After the above washing steps, the gel fragments can be stored at -20 °c for several weeks and can be reused for gel digestion.
For two-dimensional gel, it is not necessary to carry out the reduction and alkylation of cysteine residues prior to gel digestion.
(1)Place the gel fragments on ice and add 8 μl of cooled digestion buffer.
(2)After 20 min, cover the gel with a small amount of ammonium bicarbonate buffer.
(3)Incubate at 37 °c for 4 h or overnight.
4.Extraction, concentration and desalting of polypeptide fragments
Extraction of polypeptide fragments
(1)Add 20 μl TFA solution (ultrasound for 15 min), and extract the polypeptide fragment produced by trypsin digestion. Store the supernatant in a 500 μl centrifuge tube.
(2)Extract the supernatant with 20 μl of 3:2 acetonitrile/TFA solution (ultrasound for 15 min) and collect the supernatant.
Peptide concentration and desalting
(1)Concentrate the supernatant by a vacuum centrifuge to a volume of approximately 10 μl. In this step, remove acetonitrile (otherwise the peptide fragment will not be immobilized on the hydrophobic chromatography medium), and reduce the volume to the appropriate range for chromatography in the next step.
(2)Firmly secure the ZipTip on a 10 μl adjustable pipette (back pressure may be high).
(3)Wash ZipTip 5 times with 10 μl of 3:2 acetonitrile/TFA solution. Remove the cleaning solution.
(4)Balance ZipTip 5 times with 10 μl of TFA solution. Remove the cleaning solution.
(5)Instead of raising the tip of the pipette, slowly aspirate and disperse 10 μl of the concentrated supernatant at least 10 times and fix the peptide fragment.
(6)Wash the fixed peptide (desalted) 4 times with 10 μl of TFA solution. Remove the cleaning solution.
(7)Elute the peptide fragment with 2 μl of 1:1 acetonitrile/TFA solution into a 500 μl microcentrifuge tube.
MALDI-TOF Peptide Mass Fingerprint
(1)In the MALDI process, the sample is usually co-crystallized with aromatic organics. These organics are usually acidic compounds (matrix) and their main property is to absorb the ultraviolet excitation wavelength (typically the 337 nm wavelength of a nitrogen laser or the 355 nm wavelength of a three-fold Nd:YAG laser). During the desorption process, when the ultraviolet laser is emitted, the aromatic group of the matrix (existing in a large amount, exceeding 10,000:1) absorbs ultraviolet energy, causing the matrix to sublimate into a gaseous state, so as not to decompose or dissociate the sample.
(2)The ionization process is a high-concentration, non-stop expansion and cooling gas phase proton exchange process that occurs between charged matrix ions and neutral peptide fragments (ion-molecular reactions).
(3)Once desorbed and ionized, the sample in the TOF tube accelerates toward the detector, and its velocity (TOF at a fixed length flight tube) is proportional to the root mean square of m/z.
(4)The mirror (electrostatic mirror) corrects the initial kinetic energy distribution, thereby improving mass resolution and mass accuracy (isostatic ions with greater kinetic energy have larger paths in the mirror and lower at the detector focal plane) kinetic energy).
(5)Delayed or pulsed ion extraction corrects the initial spatial distribution, thus again improving resolution and mass accuracy (according to the position at which the ions start from the source to the analyzer extraction, the isostatic ions in the desorption chamber will accelerate spatial distribution).
MALDI-TOF mass spectrometry is the first choice for PMF, mainly because MALDI-TOF has many advantages.