Mass Spectrophotometry



 

Section 1: Introduction to Mass Spectrophotometry

 

Over the past decade, mass spectrometry has undergone tremendous technological improvements allowing for its application to proteins, peptides, carbohydrates, DNA, drugs, and many other biologically relevant molecules. A mass spectrometer determines the mass of a molecule by measuring the mass-to-charge ratio (m/z) of its ion. Ions are generated by inducing either the loss or gain of a charge from a neutral species. Once formed, ions are electrostatically directed into a mass analyzer where they are separated according to m/z and finally detected. The result of molecular ionization, ion separation, and ion detection is a spectrum that can provide molecular mass and even structural information.

 

Theoretical Basis for Analyzing Mass

 

According to the Lorentz force and Newton's second law in electromagnetic field, we have the following equation.

So for different mass/charge ratio, the ions in the electromagnetic field can have different trajectories.

Solving the above two equations, we get the mass to charge ratio should satisfy:

Therefore, as different molecules have different mass to charge ratio, they follow different trajectories in the electromagnetic field. Their trajectories should also depend on their initial speed and the directions of the fields, but these can be calculated as well.

There are many types of mass analyzers, using either static or dynamic fields, and magnetic or electric fields, but all operate according to this same law. Each analyzer type has its strengths and weaknesses. Many mass spectrometers use two or more mass analyzers for tandem mass spectrometry (MS/MS). A peptide sequence tag obtained by tandem mass spectrometry can be used to identify a peptide in a protein database. In addition to the more common mass analyzers listed below, there are other less common ones designed for special situations.

 

For the Time-of-flight (TOF) analyzer, it's just an electric field accelerating the ions through the same potential.By measuring the time they take to reach the detector, when the charge is known, the mass can be determined. If the particles all have the same charge, then their kinetic energies will be identical, and their velocities will depend only on their masses. Lighter ions will reach the detector first.

 

 

 

Mass Spectrum Analysis

 

==Basic peaks==

Mass spectra have several distinct sets of peaks:

 

Mass spectra first of all display the molecular ion (or parent ion) peak which is a radical cation M+. as a result of removing one electron from the molecule. Peaks with mass less that the molecular ion are the result of fragmentation of the molecule. These peaks are called daughter peaks. The peak with the highest ratio is called the base peak which is not necessary the molecular ion. Many reaction pathways exist for fragmentation but only newly formed cations will show up in the mass spectrum and not radical fragments or neutral fragments. Metastable peaks are broad peaks at non-integer mass values. These peaks result from molecular fragments with lower kinetic energy because of fragmentations taking place ahead of the ionization chamber. They are not of analytical value.

 

 

Mass Spectrometry of Proteins

 

Mass spectrometry is highly sensitive and versatile for studying proteins. Availability of genome sequence have also established it as a powerful tool for rapidly identify proteins from very complex biological samples. Advances in sample preparation methods and bioinformatics will continue to increase the scope of this technique for proteomics applications. It is useful for quantitative analysis of proteins as well as the characterisation of protein modifications. Comprehensive data about a system would provide mechanisms and regulatory events for cellular processes.

 

The two primary methods for ionization of whole proteins are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). In electrospray ionization, a liquid is pushed through a very small, charged and usually metal capillary, aerosolizing the molecules. This method is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized. MALDI relies upon a direct laser beam to trigger ionization and a matrix to both protect and facilitate vaporiziation of the biomolecule. Both methods are similar in relative softness and ions produced.

 

 

 

Once ionized, two different approaches can be implemented in characterizing the biomolecules. In the first, the intact proteins are introduced to a mass analyser. This approach is referred to as "top-down" strategy of protein analysis as the molecules remain intact. In the second, proteins are enzymatically digested into smaller peptides (~15aa) using an proteolytic agent. This is sometimes necessary as mass spectrometry often cannot resolve proteins of considerable length. Although a variety of digest agents are used, trypsin is among the most commonly used proteolytic enzymes for this application. Trypsin, a serine protease, chiefly cleaves peptide chains at the carboxyl side of the amino acids lysine and arginine, except if there is a subsequent proline. As proteins have distinct lysine, arginine, and proline composition and sequence order, cleavage at specific sites yield distinct and predictable mass spectrum profiles allowing easy identification of unknown proteins. Peptide mass fingerprinting compares these mass spectrum profiles in silico to either a database containing known protein sequences or the genome. This is often referred to as the "bottom-up" approach of protein analysis where identification at the peptide level is used to infer the existence of proteins.

 

Whole protein mass analysis is primarily conducted using either time-of-flight (TOF) MS, or Fourier transform ion cyclotron resonance (FT-ICR). These two types of instrument are preferable here because of their wide mass range, and in the case of FT-ICR, its high mass accuracy. Mass analysis of proteolytic peptides is a much more popular method of protein characterization, as cheaper instrument designs can be used for characterization. Additionally, sample preparation is easier once whole proteins have been digested into smaller peptide fragments. The most widely used instrument for peptide mass analysis is the quadrupole ion trap. Multiple stage quadrupole-time-of-flight and MALDI time-of-flight instruments also find use in this application.

 

 

Section 2: Protein Protein Interaction(PPI)

 

Introduction and motivation of studying PPI

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Protein-protein interactions affect all processes in a cell. Proteins rarely function in isolation. It has been proposed that all proteins in a given cell are connected through an extensive network, where non-covalent interactions are continuously forming and dissociating. The forces that are responsible for such interactions include electrostatic forces, hydrogen bonds , van der waals forces and hydrophobic effects . It is believed that hydrophobic effects drive protein-protein interactions, whilst hydrogen bonds and electrostatic interactions govern the specificity of the interface. Water is usually excluded from the contact region.

 

During the protein-protein interaction, the conserved domains physically interact with each other. Thus, understanding protein interactions at domain level gives detailed functional insights upon proteins that are either characterized or new discovered. However, unlike protein-protein interactions that can be discovered by some high throughput technologies such as two-hybrid systems, domain-domain interactions largely remain unknown.

 

 

Signals from the exterior of a cell are mediated to the inside of that cell by protein-protein interactions of the signalling molecules. This process, called signal transduction, plays a fundamental role in many biological processes and in many diseases (e.g. cancer). Proteins might interact for a long time to form part of a protein complex, a protein may be carrying another protein (for example, from cytoplasm to nucleus or vice versa in the case of the nuclear pore importins), or a protein may interact briefly with another protein just to modify it (for example, a protein kinase will add a phosphate to a target protein). This modification of proteins can itself change protein-protein interactions. For example, some proteins with SH2 domains only bind to other proteins when they are phosphorylated on the amino acid tyrosine. In conclusion, protein-protein interactions are of central importance for virtually every process in a living cell. Information about these interactions improves our understanding of diseases and can provide the basis for new therapeutic approaches.

 

Methods and Techniques to study PPI

 

As protein-protein interactions are so important there is a multitude of methods to detect them. Each of the approaches has its own strengths and weaknesses, especially with regard to the sensitivity and specificity of the method. A high sensitivity means that many of the interactions that occur in reality are detected by the screen. A high specificity indicates that most of the interactions detected by the screen are also occurring in reality.

 

 

 

 

 

 

 

(a figure showing the principles of Y2H method)

 

 

Section 3: Properties of Protein-Protein Interaction Networks

 

Am example of yeast protein interaction network.

 

Shown above is a yeast protein protein interaction network, the PPIs are determined by the yeast two hybrid method. The proteins that can interact with each other are linked with a line, whereas the proteins that cannot interact with each other don't have an edge between them. From the graph, we can see that there are some highly connected nodes which can connect to a large number of other proteins. But the number of proteins with only a few edges are much larger.

 

This graph only tells us approximately how many proteins interact with each other, it does not provide any information on function or structure about any particular proteins in the graph.

 

Claims of properties of PPI network

According to some popular theory, the PPI network has a "power law" behavior. That is to say, as similar with the yeast PPI graph, the probability of appearance of highly connected nodes are infrequent, while the probability of appearance of less connected nodes are highly frequent. The frequency decays exponentialy with the increase of number of edges for the protein. Some theory argues that the formation of this power law property of PPI network comes from gene duplication. During gene duplication, when a new node is duplicated, it copies all the same edges that it's mother node has, therefore making the long-lived healthy proteins eventually evolve into highly connected node in the PPI graph. Although this theory so far to a large extend is only a conjecture, it has been viewed as one of the most widely accepted explainations of PPI network.

 

 

 

Some arguments and Ambiguities

Some claim that the current data for PPI is not enough for proving that this network has a power law property. The reason is most PPI graphs cover only several hundreds of proteins, therefore in the log scale, it's only two orders of magnitude or slightly more. And usually in the end of the log-log scale plot, the tail gets noisy and fluctuate was large, so it could well be just some other kind of distribution, like poission or gaussian, because of the noise and limited scale of the PPI data so far.

 

References:

References for Mass Spectrometry:

 

 

 

References for PPI: