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Experiment-5: Sample preparation for the MALDI-TOF MS analysis
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Experiment-5: Sample Preparation for the MALDI-TOF MS Analysis
                          
OBJECTIVE:
To prepare the test and standard samples for the MALDI-TOF MS analysis
 
THEORY:
Protein identification through the MS is the one of the main step in proteomics and depends on several factors like selection the right MS instrument its sensitivity, protein detection range, choosing the right matrix depending upon the sample type. Matrix selection plays an important role in ionizing the sample ions, which are later separated and detected in MS instrument. Matrices are low molecular weight compounds, acidic and volatile in nature, with strong absorption property in UV/IR region. Different types of matrices are available in the market, with different properties and applications. Other than the matrix selection, preparation and spotting of the sample-matrix mixture on the sample plate is also very crucial, which greatly influence the quality of the peptide spectra. In this experiment we have discussed different types of matrixes; their properties and applications and various sample preparation procedures. Different steps involved in the sample preparations for MALDI-TOF MS analysis are discussed below.
 
1. Peptide Enrichment:
In-gel digested protein samples are processed further using Zip-Tip pipette tips containing C18 or C4 media for enrichment of peptides prior to MS analysis.  Zip-Tip pipette tip is a 10 µL pipette tip with a bed of chromatography media fixed at its end. It is used for concentrating and purifying peptides as well as removing salts and interfering agents. Samples are passed through activated Zip-Tips where they are captured in particular bed of chromatography media. Salts and interfering agents, detergents are washed and finally samples are eluted in a very small volume of solvent. While enrichment of samples using Zip-Tip, pipetting should be performed carefully.
    
 

Figure 1. Zip-Tip pipette tips for peptide enrichment; a zoom out figure illustrates the bed of chromatography media fixed at the end of the tip. 

  

2. Matrix selection

After processing through Zip-Tips the samples are subjected to mass-spectrometric analysis and the first step in MALDI-TOF analysis is the selection of appropriate matrix for the sample. The matrix selection mostly depends on the sample type, molecular weight of the target to be analyzed. Different types of matrix are available in the market, with different properties and various applications. Selection of suitable matrix for a specific sample is very important, which can be narrowed down depending upon the properties and functions of the matrix.
 
Properties of matrix:
  1.  Matrices are low molecular weight organic compounds having low vapour pressure and volatile nature.
  2. Most of the matrices are acidic in nature, so that it can easily excite photon and ionize the target for analysis. Few basic matrices are also available.
  3. Matrices have strong absorption abilities in UV and IR region, so that it can absorb energy from the laser source and excited to release photons for ionization.
 
 The main functions of matrix include:
  1. Matrix enhances the crystallization of analyte and leads to co-crystallization along with analyte molecules.
  2. Matrix absorbs energy from laser source and converts both matrix and analyte into gas phase.
  3. Matrix transfers the absorbed energy from the laser to analyte molecules and helps in its ionization.
The analyte molecules in the form of charged ions start moving in the MS, and later get separated from eachother followed with detection at the end.
 
  1. α-cyano-4-hydroxycinnamic acid (α-cyano): is a hard matrix, not soluble in water and well soluble in organic solvents, transfer lot of energy during desorption and ionization.
  2. Sinapinic acid: is a soft matrix, used in analysis of high mass proteins and transfers less energy, fragmentation formed are smaller very much suitable for measurement of proteins.
  3. 2,5-Dihydroxybenzoic acid (DHB): is often used for peptides, tolerant towards salts and detergents and the resolution obtained is very less as compared to the above mentioned two matrices.
 
Table 1. List of the different commercially available matrix and their applications :
 

Name of the matrix

Preparation

Excitation wavelength

Applications

α-cyano-4-hydroxycinnamic acid (α-cyano)

50% Acetonitile, 0.1% TFA in extra pure water

337nm,355 nm

Peptides less than 5000 Da, lipids and nucleic acids

 

               Sinapinic acid

 


 

30-50% acetonitrile, 0.1% TFA in deionized water

337,355,266 nm

Peptides and proteins higher than 5000 Da and sometimes also used 

 

2,5-Dihydroxybenzoic acid (DHB)

 

 

50% acetonitrile or 50% any one of the solvent such as chloroform, acetone, methanol along with water

    337,355,266 nm

Small molecules and peptides which are not ionized by other matrices

 

Trihydroxyacetophenone (THAP)

 

 

Acetonitrile along with ammonium citrate were used for making matrix with extra pure water

337 nm,355 nm

Used for small nucleotides and also used for phosphorylation studies of proteins

 

                Picolinic acid

 

 

Dissolved in ethanol and water for matrix preparation

266 nm

Generally used for nucleotides

 

  

3. Matrix preparation

During matrix preparation our main aim should be to obtain a homogenous layer of matrix crystals containing well distributed analyte solution. To obtain the best result both organic as well as aqueous solvent are used along with ultra pure water. Organic solvents enhance the solubilization of hydrophobic side chains in protein or in nucleotides and aqueous solution helps in hydrophilic region. Matrix preparation is done by mixing matrix into a suitable solvent and vortexing for few minutes to dissolve it properly. 
 
4. Sample and matrix deposition
There are many approaches for deposition of sample and matrix on MALDI plate. People perform different methods of deposition which depends on the sample concentration and individual usage flexibility.
  • In dried-droplet loading approach, mostly, sample and matrix is mixed in microcentrifuge tube, the mixture is deposited directly on to the MALDI plate and left for drying. In case if the protein loadis more this method can be proffered. Here spotting step on the plate is very easy, the analyte comes direct in contact with laser and the ionization step is very effective.
  • In droplet spotting method, sample solution is placed on top of the matrix solution over the plate, so that the mixing happens within the drop. To avoid sample losslike in dried-droplet, this method is used. In this method, mixing may be uneven in some cases the liquid may come out of the spot boundary.
  • In sandwich process, first a small amount of matrix is deposited on the plate followed by protein sample and then again matrix is added on the previously spotted mixture. In most of the cases sandwich method is used for sample and matrix deposition. The idea behind this is to make matrix excited even before the laser starts firing the analyte. As soon as the analyte are exposed to the laser they will take up the photons released from the matrix. In this method samples not remain in direct the contact of laser, less sample volume can be used.

  

 

Figure 2. Deposition of sample and matrix on MALDI plate, care should be taken during pipetting so place the liquid mixture uniformly within the spot boundary.

 

 

 

 

 

 

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