Proteomics/Protein Identification - Mass Spectrometry/Instrumentation

1. Introduction
2. Instrumentation
3. Types of Mass Spectrometry
4. Data Analysis/ Interpretation
5. Applications for mass spectrometry
6. Databases

Instrumentation

Previous Section: Introduction

How does a Mass Spectrometer work?

A mass spectrometer is made up of three components: an ion source, mass analyzer, and a detector. The unknown sample which may originate as solid, liquid, solution or vapor, is presented to the ionization source. After ionizing the sample, the ions of the sample are passed to the mass analyzer region where separation based on the mass-to-charge ratio occurs. Once separated by the analyzer, the ions then enter the detector portion of the mass spectrometer. At this point, the machine calculates the mass-to-charge ratio and the relative abundance of each of the different ions. From this information, a spectrum graph can be created such as the one to the right.

Most mass spectrometers are maintained under a vacuum to improve the chances of ions traveling from ionization source to detector without interference by collision with air molecules.

Ion Source

The ion source is the mass spectrometer component which ionizes the sample to be analyzed. Ionization mainly serves to present the sample as vaporized ions which can be acted upon by the mass analyzer and measured by the ion detector. There are many different methods available to ionize samples, such as positive or negative ion modes. The ionization method chosen should depend on the type of sample and the type of mass spectrometer.

Ionization Methods:

File:ES-MassSpec.jpeg

Electronspray ionization

MALDI

There are two types of ionization methods, electron and chemical. Electron ionization involves application of an electrical current to the sample to induce ionization. Chemical ionization involves interaction of the sample with reagent molecules to induce ionization. Ions produced are often denoted with symbols that indicate the nature of the ionization, eg. [M+H]+ is used to represent a molecule which is protonated.

• Electron ionization
  
  • Electrospray Ionization
    
  • Field Desorption / Field Ionization
    
• Chemical Ionization
  
  • Matrix Assisted Laser Desorption Ionization
    
  • Fast Atom Bombardment
    
  • Thermal Ionisation
    

The methods commonly used in proteomics are ‘Matrix Assisted Laser Desorption Ionization’ or MALDI and ‘Electrospray Ionization’ also known as ESI.

MALDI uses a solid support target plate where a UV active matrix (solid or liquid) is spotted on the plate followed by the sample over the matrix. The laser hits the spot on the crystallized matrix and transfers energy from the matrix molecule to the sample. This energy transfer vaporizes the sample, sending a plume of ions into the MALDI source. This plume of ions is then collected and held in the source until a pulse sends them all out simultaneously. If the MALDI is attached to a Time of Flight(TOF) mass analyzer these ions are then sent down the TOF tube (typically ~2m) and are separated according to their velocity (light ions hitting first). MALDI is the preferred instrumentation for proteomics due to ease of reading the spectrum; most ions are found in the +1 charge state [M+H].

Electrospray, on the other hand, is done by injecting the sample dissolved in a slightly acidic solution through a heated capillary that has a voltage around 10v. This allows for highly charged particles to be formed at the tip of the capillary. As the particles evaporate, their charge/volume increases to a point where charge repulsion forces take over and the particle will explode. A small drop will form which continues the process until individual molecules are in the gas phase and charged. These ions will then travel into the analyzer, typically a quadrupole, to be scanned one mass at a time. The molecules in electrospray tend to be multiply charged and even though the upper mass limit of a quadrupole is 2000 m/z, the multiple charges allow for high mass ions or proteins to be identified. Proteins will have a charge envelope in which each peak has a different amount of charges on them. Special software is needed to deconvolve the multiple charge species peaks into a single mass peak, such as MassLynx from Waters.

Mass Analyzers

The mass analyzer is the component that separates the ions created from the ion source by their mass-to-charge ratios. Mass analyzers are based on the principles of charged particles in an electric or magnetic field. By using Lorentz force law and Newton’s second law of motion you can generate the following equation:

    $(\frac{m}{q})𝐚=𝐄+𝐯\times 𝐁$

Where m is the mass, the ionic charge is q , a the acceleration, E is the electric field, and the vector cross product of the ion velocity and the magnetic field is v x B. This equation says that two particles with the same mass to charge ratio (m/q) will behave exactly the same.

So what this equation is basically saying is that the mass to charge ratio acts as a determinant of acceleration of the ion, which can also be represented as the addition of the electric field plus the cross product of the ion velocity and magnetic field.

Scanning Mass Analyzers

Scanning mass analyzers need to separate the ions based on their relative abundances and mass to charge ratios. Electromagnetic fields are used to separate the ions based on their mass to charge ratios, by using a slit they are able to regulate which mass to charge ratio ions get to the detector. Once selected for a particular mass to charge ratio, the ion current is then recorded as a function of time which is analagous to mass.

Quadrupole Mass Spectrometers

You can think of this method as a filter or funnel which only allows certain ion masses to pass through. The "funnel" is actually a combination of positively and negatively charged metal rods which together form a channel through which the ions travel. The theory is that only selected masses will be able to pass through the channel, as all other ions won't have a stable trajectory though it and will hit the quadrupole rods, stopping the ion from reaching the detector.

Time of Flight Mass Analyzers

The TOF (Time of Flight) is a mass analyzer that allows ions to flow down a field free region; which allows the ions with a greater velocity, lighter ions, to hit the detector first. This is especially compatible with MALDI due to the fact that the TOF needs a pulsed instrument for its source. In this way ions are generated in the MALDI source and held there for a brief time and all are pulsed into the TOF at the same exact time. In this way, If all ions have the same kinetic energy, the ions with the lower mass will have a higher velocity and reach the detector first; whereas the ions with the higher mass will have a lower velocity and hit the detector last.

The kinetic energy of an ion leaving a source if given by:

    $T=eV=\frac{m{v}^2}{2}$

Where velocity v is defined by the Length of the path L divided by time t.

    $v=\frac{L}{t}$

By substituting this equation into the first and solving for time you arrive at.

    $t=L*\sqrt{(\frac{m}{e})*(\frac{1}{2V})}$

From this equation you can easily see how mass directly affects travel time. Mass is directly proportional to time. Using the m/e portion of the equation you can clearly see how a larger mass means a longer time, and likewise how a lower mass would mean a smaller m/e and thus shorter travel time.

Ion Cyclotron Resonance Spectrometer

ICR is a form of trapped Ion mass analyzer, that specifically is defined as a static trap. It is basically a box with three parallel metal sides. Trapped ion analyzers work by keeping the ions in the trap and controlling the ions by using positive and negatively charged electrical fields in a carefully series of timed events.

ICR specifically works on the principle that in a magnetic field, ions move in a circular path whose frequency is mass dependent. So using the cyclotron frequency you can surmise the mass. Equating the Lorentz force in a magnetic field to the equation for centripetal force yields.

    $evB=\frac{m{v}^2}{r}$

You can then easily solve the above equation for the frequency F or

    $F=\frac{v}{r}$

Groups of ions with the same mass to charge ratios will have the same cyclotron frequency but may be moving out of synch with one another, this is why an excitation pulse is needed to bring the resonant ions into phase with one another and the excitation pulse. Next the ions which will be passing close to the ICR cell receiver plates cause "image currents" which can be collected and amplified and analyzed. This signal registered in the receiver plates depends both on the number of ions and their distances from the receiver plates.

Detector

Those ions which pass through analyzer are now separated by the desired methods. This mass spectrometry component records the charge induced by an ion passing by a surface or current produced when an ion hits a surface. From these charges or currents, a mass spectrum can be produced as well as measure the total number of ions at each each m/z which are present. Due to the fact that the number of ions entering the detector at any given moment is minuscule, signal amplification is often necessary.

Next section: Types of Mass Spectrometry

References

• Experimental Techniques: MALDI TOF, http://www.microbiology.science.ru.nl/tech/malditof/
  
• An Introduction to Mass Spectrometry, http://www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htm
  
• Electron Ionization Sources: The Basics, http://www.spectroscopymag.com/spectroscopy/article/articleDetail.jsp?id=358670
  
• What other techniques are used to produce ions?, http://www.asms.org/whatisms/p11.html
  
• Fourier Transform Ion Cyclotron Resonance theory: a brief review, http://www.emsl.pnl.gov/docs/msd/mass_spec/home/fticrtut.html