Principles and Methods of Mass Spectrometry Analysis of Proteins

Mass spectrometry is sensitivity and accurate, and can accurately identify proteins. At present, mass spectrometry mainly identifies the primary structure of egg, including molecular weight, amino acid sequence of peptide chain and number and position of polypeptide or disulfide bond, which plays an important role in the study of protein structure analysis.

Mass spectrometry consists of injector, ion source, mass analyzer, ion detector, control computer, and data analysis system. Traditional mass spectrometry is only used for the analysis of small molecule volatiles, but with the emergence of new ionization techniques such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization mass spectrometry (ESI-MS), etc. The emergence of various mass spectrometry techniques provides a new and accurate way for protein analysis. Now, the combined application of enzymatic hydrolysis, liquid chromatography separation, tandem mass spectrometry and computer algorithms has become the development trend of protein identification.

The basic principle of mass spectrometry analysis of proteins is to convert protein molecules into ions by ionization source, and then use the electric field and magnetic field of the mass spectrometer to separate protein ions with specific mass and charge ratio (M/Z), and pass the ion detector. The separated ions are collected, the M/Z value of the ions is determined, and the unknown protein is analyzed and identified. Proteins can be identified more accurately and quickly, often in conjunction with appropriate processing and other techniques.

Methods of Protein Mass Spectrometry

There are three main methods for mass spectrometric analysis of proteins: peptide mass fingerprinting (PMF), tandem mass spectrometry (CID), and ladder peptide sequencing.

Peptide Mass Fingerprinting (PMF) 

Peptide mass fingerprinting (PMF) uses a specific enzymatic or chemical hydrolysis method to cut the protein into small fragments, and then uses mass spectrometry to detect the relative molecular mass of each product peptide, and the mass of the obtained proteolytic peptide is corresponding. The database is searched for similar peptide fingerprints to draw a "peptide map". It can be seen that the accuracy of molecular mass is the key indicator of PMF. While the post-translational modification of protein may make the mass of PMF inconsistent with the theoretical value, so it needs to be properly combined with the sequence information.

Tandem Mass Spectrometry (CID)　　

Tandem mass spectrometry (CID) uses the metastable ions generated by the molecules measured during ionization and flight to identify the corresponding amino acid residues by analyzing the mass difference of adjacent peaks of the same type. The peptide sequence map of tandem mass spectrometry requires the reading of a partial amino acid sequence in combination with the ionic mass before and after the peptide mass. This method of identification is called peptide sequence tag (PST).

Ladder Peptide Sequencing

The Ladder peptide sequencing is similar to Edman method in that a chemical probe or enzymatic hydrolysis is used to degrade the amino acid residue from the N-terminus or the C-terminus, resulting in a series of peptides containing only one amino acid residue. It is called ladder, and it is detected by mass spectrometry, and the corresponding amino acid residues are known from the difference in mass of adjacent peptide peaks. The problem is that the rate of enzymatic hydrolysis is different and it is susceptible to interference.

Problems in Protein Mass Spectrometry　　

l Problems about Protein Digestion

The larger the group of protein-digested proteins, the lower the accuracy of mass spectrometry detection. Therefore, before mass spectrometry, the protein must be digested into small molecule peptides to improve the accuracy of mass spectrometry. In general, peptides of 6-20 amino acids are most suitable for the detection of mass spectrometers. The most commonly used enzyme today is trypsin, which cleaves the lysine and arginine of the protein. Therefore, the same protein will be produced by trypsin digestion.

l Problems about Protein Modification

It is difficult to give 100% of the sequence of the peptide under current conditions. Some peptides are often lost. Protein phosphorylation also inhibits enzymatic hydrolysis of trypsin, and the amount of phosphorylated peptide is much less than that of non-phosphorylated peptides, therefore the response of mass spectrometry to phosphorylated peptides may be inhibited. Thus, the content of non-phosphorylated peptides should be minimized as much as possible. Methods for reducing non-phosphorylated peptides include fractionation, IMAC (solid phase metal affinity chromatography) and antibody binding.

l Problems about Accuracy

In mass spectrometry sequencing, the accuracy of mass spectrometry has a great impact on the results of the assay, since the key to mass spectrometry is to determine the molecular weight difference between adjacent peptide chains, as to determine the corresponding amino acid residues. If the measured absolute mass error is 0.5 or more, it is impossible to effectively distinguish amino acid residues with a mass difference of 1 or less.

Instrument reference


l Q Exactive Focus Combined Quadrupole Orbitrap Mass Spectrometer

Comments

Post a Comment

Popular posts from this blog

Practice of N-terminal Sequencing by Edman Degradation Technology in Protein and Peptide Sequence Analysis

Image
The N-terminal amino acid sequence of protein is one of the key quality attributes of biological drugs. N-terminal sequencing by Edman degradation  is a routine method to analyze this property. The analysis of 15 amino acid sequences on the N-terminal by Edman degradation method is usually a necessary item when applying for biological drugs . At  the same time, it is also an annual inspection item of many listed biological drugs (see Pharmacopoeia 2015). In scientific research, N-terminal sequencing can be used to provide key information for the confirmation of unknown or uncertain theoretical sequences. So this method is widely used in scientific research and industry.  W hat are its principles ? H ow to analyze the results ? Advice for sample delivery? P rinciples In short, we can understand it from these two aspects: The first step  is Edman degradation . T hrough the reaction of PITC (Phenylisothiocyanate), an organic reagent, and α - amino acid at the N-terminal of pr
Read more

Part I: Basic Knowledge of Quantitative Analysis of Protein Acetylation Modification

Image
After the protein is translated in the cell, it is processed through a very important step before being transported to the corresponding organelle and undergoing specific biological effects. The role of protein   post-translational modification   ( PTM ) is primarily to alter the activity, localization or function of the protein. Protein PTM  also further increases the diversity and complexity of cellular pathway mechanisms and life activities. Except for acetylation , c ommon protein PTM  include s   phosphorylation , glycosylation , ubiquitination , and the like. Protein  A cetylation  M odification Protein acetylation modification, as the name suggests, refers to the grafting of acetylated groups on the original basis of the protein. In cells, the acetylation modification reaction is catalyzed by an acetyltransferase, and the acetyl group of acetyl-CoA is transferred and added to the protein lysine residue. In earlier studies, acetylation has been considered to be a pos
Read more

De Novo Protein Sequencing Procedures and Features

Image
The sequence of a protein molecule is the basis for the function of a protein. Accurate determinat ion of protein sequence plays an important role in the study of protein functional epitopes, detection of commercial monoclonal antibodies, vaccines, and kits. Although there are many studies on protein sequencing, accurate sequencing of proteins is still not as fast and accurate as that of DNA. Protein sequencing is mainly hampered by the purification of protein samples, and the complexity of factors that make up the amino acid com position of proteins. De novo   protein sequencing  is  also called novel protein sequencing. This technique deduces t he amino acid sequence based on the difference in mass between a series of regular fragment ions generated by the peptide collision with an inert gas. We can guess the amino acid sequence and post-translational modifications based on the y ion and b ion at the cleavage of the peptide bond. De novo  sequencing has a unique advantage that t
Read more