Peptide mass fingerprinting and protein identification by MALDI-TOF method
MALDI-TOF peptide mass fingerprinting (PMF) is a faster and more convenient method for identifying proteins. The genomes studied have been sequenced and annotated, and proteins can be separated and detected by two-dimensional gel electrophoresis. The source of this experiment is "Guide to Plant Proteomics Experiments" [France] H. Tillemment, M. Zivi, C. Damerweil, V. Mitschine, eds.
Operation method
Peptide mass fingerprinting-MALDI-TOF method for protein identification
Materials and Instruments
Sequencing Grade Porcine Trypsin Move Many gel staining methods are suitable for subsequent MALDI-TOF peptide mass fingerprinting (PMF ) analysis. The performance and compatibility of some staining methods have been discussed in Chapter 14. For more product details, please visit Aladdin Scientific website.
Ammonium bicarbonate Ammonium bicarbonate buffer Acetonitrile Ammonium bicarbonate buffer Digestion buffer
Vacuum centrifuge Oven
3.1 In-gel digestion
The in-gel digestion method was adapted from Jensen et al.
For the gel handling and digestion steps, samples must be handled carefully to minimize exogenous keratin contamination (e.g., hair, skin, woolen clothing, air currents, ventilation cooling, etc.) (see Note 1).
1. Digging protein spots from 2D polyacrylamide gels
( 1 ) Make a perforator by excising the 1 ml tip of the gun with a razor blade at 5 mm from the tip (incision diameter 1 to 2 mm) ( see Note 2).
( 2 ) Scoop the gel spot and transfer to a centrifuge tube.
2. Wash gel fragments
( 1 ) Wash with 100 μl ammonium bicarbonate buffer (shaking for 15 min), then discard the supernatant.
( 2 ) Wash twice with 100 μl acetonitrile/ammonium bicarbonate buffer (shaking for 15 min) and discard the supernatant.
( 3 ) Wash with 100 μl HPLC grade acetonitrile (shaking for 15 min) to shrink the gel fragments, and then discard the supernatant.
( 4 ) Vacuum dry the gel fragments on a centrifugal evaporator.
After these washes, the gel fragments can be stored at -20°C for several weeks and can be reused for gel digestion.
For two-dimensional gels, reduction and alkylation of cysteine residues prior to gel digestion is not necessary (see Note 3).
3. In-gel digestion (see Notes 4 and 5)
( 1 ) Place the gel pieces on ice (see Note 6) and add 8 μl of cooled digestion buffer.
( 2 ) After 20 min, adjust with a small amount of ammonium bicarbonate buffer to cover the gel.
( 3 ) Incubate at 37°C for 4 h (or overnight, see Note 7).
4. Extraction, concentration and desalting of peptide fragments
1 ) Extraction of peptide fragments
( 1 ) Add 20 μl of TFA solution (sonication for 15 min) to extract the peptide fragments produced by trypsin digestion. The supernatant was stored in a 500 μl centrifuge tube.
( 2 ) Extract the peptide fragments with 20 μl of 3:2 acetonitrile/TFA solution (sonication for 15 min). The supernatant was collected.
2 ) Peptide concentration and desalting
( 1 ) Concentrate the supernatant to a volume of about 10 μl by vacuum centrifugation. This step removes acetonitrile (otherwise the peptide fragments will not be immobilized in the hydrophobic chromatographic medium) and reduces the volume to a suitable range to allow for the next chromatographic step.
( 2 ) Secure the ZipTip firmly to a 10 μl adjustable pipette (back pressure may be high).
( 3 ) Wash the ZipTip 5 times with 10 μl of 3 : 2 acetonitrile/TFA solution. Remove the wash solution.
( 4 ) Equilibrate the ZipTip 5 times with 10 μl of TFA solution. Remove the cleaning solution.
( 5 ) Without raising the pipette tip, slowly aspirate and disperse 10 μl of concentrated supernatant 10 times to immobilize the peptide fragment.
( 6 ) Wash the immobilized peptide (desalted) 4 times with 10 μl of TFA solution. Remove the washing solution.
( 7 ) Elute the peptide fragments with 2 μl of 1:1 acetonitrile/TFA solution into a 500 μl microcentrifuge tube.
3.2 MALDI-TOF peptide mass fingerprinting
( 1 ) In the MALDI process, samples are usually co-crystallized with aromatic organics. These organics are usually acidic compounds (matrices), and their main property is that they absorb the UV excitation wavelength (typically 337 nm for nitrogen lasers or 355 nm for triple-frequency Nd: YAG lasers). During desorption, when the UV laser is emitted, the aromatic groups of the matrix (which are present in large quantities, more than 10,000 : 1 ) absorb the UV energy, causing the matrix to sublimate into a gaseous state without decomposing or dissociating the sample.
( 2 ) The ionization process is a highly concentrated, non-stop expanding and cooling gas-phase proton exchange process occurring between charged matrix ions and neutral peptide fragments (ion-molecule reaction).
( 3 ) - Upon desorption and ionization, the sample in the TOF tube accelerates toward the detector, and its velocity (in a fixed-length flight tube of TOF) is proportional to the root mean square of m/z.
( 4 ) The mirror (electrostatic mirror) corrects the initial kinetic energy distribution, thus improving mass resolution and mass accuracy (isobaric ions with higher kinetic energies have larger paths in the mirror and lower kinetic energies in the focusing plane of the detector).
( 5 ) Extraction of delayed or pulsed ions corrects the initial spatial distribution and thus again improves resolution and mass accuracy (depending on the position of the ions from the source to the beginning of the analyzer extraction, the isobaric ions in the desorption chamber will accelerate the spatial distribution).
A number of specialized literature or books covering the basics of MALDI-TOF and MS are now available, see them as well.
MALDI-TOF mass spectrometry is the preferred technique for PMF, mainly because of a number of advantageous features.
( 1 ) Soft ionization (peptide fragments remain intact during the MALDI process).
( 2 ) The ionization method is also suitable for the analysis of different peptide mixtures, and the method is more resistant to contamination (buffers, salts, plasticizers, etc.).
( 3 ) The method is highly sensitive (MALDI process and TOF mass analyzer available).
( 4 ) Good resolution, currently over 15000 [ full width, half mass (FWHM) ], with detection of single isotope peaks in the mass range of interest.
( 5 ) TOF analyzers have high mass accuracy (typically 10-30 ppm).
( 6 ) It can get the experimental data quickly.
1. Target sample deposition
1 ) Classical dry droplet method
( 1 ) Preparation of semi-saturated cyano-4-hydroxycinnamic acid (approx. 5 mg/ml): dissolve α-cyano-4-hydroxybenzoic acid in 300 μl of 1 : 1 acetonitrile/TFA solution (sonicate for 10 min). The supernatant was centrifuged for a few seconds. α-Cyano-4-hydroxycinnamic acid was prepared by adding 200 μl of the supernatant to a microcentrifuge tube and adding the same volume (200 μl) of 1:1 acetonitrile/TFA solution to obtain a semi-saturated form of α-cyano-4-hydroxycinnamic acid.
( 2 ) Take 0.8 μl of Digestion Solution and 0.8 μl of Substrate and add to a microcentrifuge tube and mix quickly (avoid crystallization at the pipette tip). Take 0.8 μl of the mixture and drop it onto the MALDI substrate, the remaining 0.8 μl of the mixture can be dropped at another location on the MALDI substrate. The pipette tip should not touch the substrate.
( 3 ) Allow the mixture to dry and crystallize naturally (see Note 8).
2 ) Dry the droplet on the hydrophobic substrate (see Note 9).
( 1 ) Prepare α-cyano-4-hydroxycinnamic acid: weigh 10 mg of α-cyano-4-hydroxycinnamic acid and dissolve in 1 ml of 1:1 acetonitrile/TFA solution (sonicate for 10 min). Take 56 μl of the above solution into a new microcentrifuge tube and add 994 μl of 1:1 acetonitrile/TFA solution.
( 2 ) Rapidly mix 0.8 μl of gel-digested supernatant (see Section 19.3.1 3 ) with 0.8 μl of the above solution.
Take 0.8 μl of the mixture and drop it onto the MALDI substrate, the remaining 0.8 μl can be dropped onto another part of the MALDI substrate [see Section 19.3.2 1.1)].
( 3 ) Allow the mixture to dry and crystallize naturally.
( 4 ) Add 4 μl of TFA solution dropwise onto the crystals, and after 30 s of contact, remove the liquid with a pipette (the tip of the pipette should not touch the crystals).
( 5 ) Add 0.8 μl of ethanol/acetone/TFA solution and crystallize again.
( 6 ) Let the mixture dry naturally and crystallize out.
2. Spectral acquisition
( 1 ) Insert the substrate into the mass spectrometer and stabilize it in vacuum (below 10-6 Torr).
( 2 ) Turn on the high voltage [it is better to wait 20 min for the voltage and temperature to be constant (Joule effect)].
( 3 ) Adjust the laser power (attenuation) and the position of the base to obtain a good signal-to-noise ratio and single isotope resolution at 700~4000 Th.
( 4 ) The laser is fired 10 times, but the resulting maps are of little value because they are usually noisy maps of small mass molecules (substrates and/or salts).
( 5 ) Laser beams are fired 80 to 200 times, and the maps are acquired and superimposed.
( 6 ) The mass spectrum was internally corrected with ions obtained by hydrolyzing trypsin at 842.5099 Th and 2211.1046 Th.
( 7 ) Save the spectrum (see Note 10).
3. Spectral analysis
Automatic designation of single isotope masses is possible, but we recommend that these resolutions should be scrutinized (see Remark 11 and Remark 12).
If the plots are not smoothed, all initial single-isotope peptide peaks can be resolved, except for those known to be produced by trypsin (Figure 19-1, see Note 13 and Note 14 for detailed descriptions). 
3.3 Evaluation of MASCOT Database Search Use and Search Results
The steps described below were performed against the MASCOT PMF search engine (Matrix Science, London, UK ) licensed internally (see Note 15 ) or using a server located as far away as London (http://www.matrixscience.com/cgi/search_ form, pi? SEARCH = PMF) The
Of course, the described search strategy is directly applicable to other search engines (see note 16).
1. First steps of the search
The purpose of this step (left side of Fig. 19-2) is to obtain a general picture of the quality of the data in order to eliminate possible contaminants and potentially confirm plant proteins directly.
( 1 ) Identify a globally open database (e.g., MSDB, NCBInr, or SWISS- PROT), rather than one of the species-specific ones, that contains protein contaminants that are not plants.
( 2 ) Do not specify species in the taxonomic domain (i.e., " all items" ) to identify non-plant protein contaminants.
( 3 ) Allow a gap to be missed.
( 4 ) Set a large mass tolerance value of 100 ppm to evaluate mass spectrometry calibration ( two trypsin-hydrolyzed peptides used for mass spectrometry calibration may have low abundance and ion statistical significance, and do not calibrate well ) .
( 5 ) Immobilization modifications: carboxymethyl (C) or ureidomethyl (C). Reduction and alkylation of protein cysteine residues of gel samples with iodoacetic acid or iodoacetamide produces carboxymethylcysteine or amidomethylcysteine (included in ureidomethylcysteine), respectively. 
( 6 ) Modification of variables: none.
Desired protein information can be obtained if the data results are good (good signal-to-noise ratio, high mass accuracy, few contaminants) and if the database can be searched for reference proteins (Fig. 19-2B ).
Candidate proteins are evaluated according to the following criteria:
( 1 ) Score better than the important critical value.
( 2 ) The difference between the most optimal protein and the unrelated proteins is large.
( 3 ) The protein of the studied species (or a similar species with high homology) is the first level of the optimal protein.
( 4 ) The molecular masses and isoelectric points of the candidate proteins are compatible with the two-dimensional gel data.
( 5 ) Good mass accuracy (best) or bad mass accuracy (due to calibration failures or mis-calibration problems ), but in the mass range with a low and linearly distributed correlation with mass dispersion.
( 6 ) - Maximization of one cleaved peptide over three matching peptides.
( 7 ) Minimization of five matching peptides.
( 8 ) Homogeneous localization of matching peptides in protein sequences and sequence coverage (see Note 17 and Fig. 19-2C).
2. Second and subsequent search steps
If the search yields no results, or if too many peaks (e.g., more than 10) do not match (which may indicate that the dug site contains two or more proteins) (see Note 18 and the right-hand side of Fig. 19-2), further searches are required.
Additional search criteria can be selected (Fig. 19-2D):
( 1 ) Remove the Digestion Contaminant Peak that identifies a non-vegetable peptide ("Search Unrivaled" Mascot Options button ).
( 2) Set the mass tolerance based on the actual observed mass accuracy of the mass spectrum (e.g., as assessed for non-plant protein contaminants or as verified with respect to trypsin auto-digested fragments). Typically, good quality searches require a mass accuracy of less than 30 ppm (lower values are better).
( 3 ) Searches with non-stoichiometric or unpredictable chemical modifications (mainly methionine oxidation, methyl esterification of aspartate and glutamate, and N-terminal pyroglutamylation) are permitted (see Note 19).
( 4 ) Limit the scope of the search to taxa (e.g. Viridiplantae protein database).
( 5 ) Allows searching in a specific database.
The evaluation and confirmation criteria described in Subsection 3.3.1 are still necessary (Figures 3-2E and F). It is also important to identify the secondary components of the mixture.
After a protein has been successfully identified by peptide mass fingerprinting (PMF), there are often several ion peaks that are not identified by MALDI-TOF mass spectrometry. These unidentified peaks may have different origins:
( 1 ) Peptide ion peaks of other proteins that co-migrate in the electrophoresis gel.
( 2 ) Peptide ion peaks of the same protein, but with minor differences between its amino acid sequence and the protein sequence in the search database (possibly caused by genomic annotation errors or mutations).
( 3 ) A peptide ion peak for the same protein, but its molecular mass does not match the predicted peptide mass due to post-translational modifications.
( 4 ) Peptide ion peaks that appear due to protein contamination (e.g., keratin or tryptic protein).
( 5 ) Non-peptide ion peaks due to polymer residue or plasticizer contamination.
( 6 ) Non-peptide ion peaks due to biochemical reagent drug contamination.
3.4 MALDI-TOF/TOF strategy
The proteins identified by PMF are only used as preliminary candidates, and the score of the identified proteins indicates the degree of confidence. Therefore, we should further perform secondary sequencing of the ion peaks obtained by PMF to improve and confirm the confidence of the identified proteins.
Before the recent invention of TOF/TOF tandem mass spectrometry analyzers, the success rate of sequencing peptides on MALDI mass spectrometry analyzers was not high. On a TOF/TOF analyzer, a primary TOF separates the peptide ions we need, and the other peptide ions are directly removed, and the remaining peptides are specific amino acid sequence peptides after screening by bombardment with a primary TOF. Then the secondary TOF separates and measures the molecular mass of these specific amino acid sequence fragments.
1. Identification Confirmation
To confirm the confidence of the identification, 1~3 peptides identified from a single protein are successively bombarded by TOF/TOF. The resulting fragment ions must match the predicted peptide sequence.
2. Identification of unknown proteins
In the case of unsatisfactory identification by protein databases (see 3.3), some peptides (usually 2-5) can be analyzed by TOF/TOF. Ab initio sequencing combined with a protein database search for TOF/TOF ion fragments of these peptide ions may be successful in identifying these unknown proteins. The database selected for identification should be the protein database of the taxon to which the subject belongs or of a species with interspecies cross-correlation (see LCMS/MS section in Chapter 20).