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Mass Spectrometry

Principle

A mass spectrometer is an analytical tool used to measure the molecular mass of a sample. The three fundamental parts of a mass spectrometer are the ionization source, analyzer and the detector. A mass spectrometer does the following:

  • Produces ions from the sample in the ionization source.
  • Separates these ions according to their mass-to-charge ratio in the mass analyzer.
  • Fragmentizes the selected ions and analyzes the fragments in a second analyser.
  • Detects the ions emerging from the last analyser and measures their abundance with the detector that converts the ions into electrical signals.
  • Processes the signals from the detector that are transmitted to the computer and controls the instrument through feedback.

MALDI

There are a variety of ionization methods available, however the most commonly used methods are: Electrospray Ionization (EI) and Matrix-assisted Laser Desorption Ionization (MALDI). In this lab we use MALDI.

There are two major steps involved in MALDI, which uses a UV-laser to ionize a sample. First, the peptide to be analyzed is dissolved in a matrix, which is a solvent containing small organic molecules. These molecules have high absorption at the laser’s wavelength. The mixture is then left to dry prior to analysis. This results in the formation of a crystal matrix that contains the peptide of interest. Second, the matrix molecule is excited to a higher energy state when it encounters the UV-laser. This eventually leads to the formation of the ion of interest. The ionized molecule enters the mass analyzer and yields the mass spectrum.

TOF

The MALDI ionization method is coupled with a mass analyzer called time-of-flight (TOF). In TOF, the time ions take to reach the detector is measured. The velocity is determined by the mass-to-charge ratio (m/z). Smaller ions will reach the detector before large ones due to the fact they have less mass. The charge of the ions also determines the velocity. Ions with 2 or more positive charge will move faster than ions with only 1 positive charge. Thus, those ions that are both smaller and positively charged will move faster.

The output of the detector is the mass spectrum, displayed in a “stick diagram”. This shows the relative current produced by ions of varying mass/charge ratios.

Schematic diagram of a mass spectrometer. At the bottom left, a laser is directed at a target plate where the sample is placed. The laser light passes through a lens system, shown by two red lines converging, before hitting the target plate. Below the target plate, there's an optical sensor. In the center of the image, the ionized sample particles are represented by a large arrow pointing upwards, indicating their movement toward the detectors. At the top, there is a detector directly aligned with the path of the ions, and a reflectron to the right, which is used to reflect ions to a post-acceleration detector located on the left-hand side. The post-acceleration detector is labeled and has horizontal lines indicating the detection plates. On the bottom right, there's a label for an X-Y stage, which moves the target plate to allow for scanning of different areas.

Figure 1. Mass Spectrometer

Detection of Protein

Proteins are linear polymers made up of combinations of the 20 most common amino acids linked to each other by peptide bonds. The protein produced by ribosomes undergoes covalent modification called post-translation modification. Over 200 modifications have been identified.

Detection of rhEPO R H E P O is conducted using a mass spectrometer. The mass-to-charge ratio (m/z) is roughly the same as the protein mass in Dalton. We use a urine sample for substance detection. In most cases, it is better to use a urine sample instead of blood to test for prohibited substances. This is because urine collection is non-invasive and yields a large sample volume with a higher drug concentrations than blood. Also, urine has fewer cells and proteins that would complicate extraction.

To detect prohibited substances in urine, we have to first make a standard spectrum of uncontaminated urine (negative standard), as well as of the prohibited substance (positive standard). Once we have these two standards, we can compare them with a sample spectrum. If the sample spectrum exhibits the same peaks that the positive standard has, we can conclude that the sample contains the prohibited substance. Mass spectrometry can also detect glycosylation, as the data from the mass spectrometer is processed and displayed in peaks. Usually, glycosylation peaks are marked with an asterisc or a diagram of glycan.

Synthesis of Protein

Lab Biosafety

Theory overview

Referred from:

Article
Protein Synthesis
Article
Lab Biosafety