Question

5. An unknown human protein was digested with the enzyme trypsin and the mixture of the resulting tryptic peptides were analysed by MALDI-TOF/TOF mass spectrometry. A TOF MS spectrum of the peptide mixture and three MS/MS fragmentation spectra from the three most intense peptide ions have been collected. Explain how this protein could be identified.

Answer 1

MALDI-TOF/TOF Mass Spectrometer

It is a tool that has been instrumental in the protein revolution, which features a MALDI (matrix-assisted laser desorption ionization) source and tandem time-of-flight (TOF/TOF) mass analyzers. It has the ability to deconvolute highly complex mixtures over a wide range of abundance levels. It helps to identify and quantify proteins and to determine if and how those proteins have been post-translationally modified. The spectrometer measures an ion's mass-to-charge (m/z) ratio. It helps in peptide mass fingerprinting, which is the identification of a protein based on the specific group of peptide masses it produces. Apart from these the MS/MS instruments, can provide peptide sequence information as well.

STEP 1

The underlined process typically begins either with a protein spot which is extracted from a 1-D or 2-D protein gel, or with liquid chromatography fractions. The proteins are all enzymatically treated (e.g., with trypsin). It is then mixed with matrix (typically alpha-cyano-4-hydroxycinnamic acid or dihydrobenzoic acid), arrayed on a metal target plate, and allowed to dry. The plates enable high-throughput research which helps in several hundred samples at once. Equipping the instrument with an autoloader increases the level of walk-away automation.

STEP 2

Once an X-Y translation stage has positioned a particular spot, an ultraviolet laser (a nitrogen laser firing at 337 nm or a Nd:YAG laser at 355 nm, for instance) strikes the sample. The matrix material absorbs this very light energy, generating enough heat to vaporize and ionize the peptide sample, generally with a charge of +1.

STEP 3

Just as the ion races down the flight tube, its speed is a function of its m/z ratio. Hence, a particle's m/z ratio is measured based upon its time of flight.

STEP 4

In tandem MS (MS/MS) mode, the ions pass through a gauntlet of components that select specific ions for further analysis, slow them down, fragment them, and then re-accelerate them. We have to keep in mind that in standard MS mode, these four components are not used. Of the two ions shown (blue and green) in the original sample, only the blue ions are selected for fragmentation, known as collision-induced dissociation (CID) analysis.

STEP 5

Mass resolution improves with the length of the flight path, so many TOF (time-of-flight) mass spectrometers contain a reflectron, which functionally elongates the tube without physically doing so. The reflectron also serves to energy-focus ions, correcting their velocity spread to further improve resolution. For simple applications, however, users can forego the reflectron and use the linear detector instead.

STEP 6

These detectors measure each ion collision, producing a graph, or spectrum, of m/z versus intensity.

STEP 7

Comparing the resulting constellation of m/z values against a database identifies the peptide, and by extension, the protein from which it derives. Tandem MS/MS analysis of fragmented peptides produces an actual sequence, providing a more accurate protein ID. Today's mass spectrometers have high enough resolution and mass accuracy to distinguish peptides bearing only subtle differences, resulting in confident and thorough identifications of proteins from extremely complex mixtures.

Time-of-flight mass spectrometry (TOFMS) is a method of mass spectrometry in which an ion's mass-to-charge ratio is determined via a time of flight measurement. Ions are accelerated by an electric field of known strength.^[1] This acceleration results in an ion having the same kinetic energy as any other ion that has the same charge. The velocity of the ion depends on the mass-to-charge ratio (heavier ions of the same charge reach lower speeds, although ions with higher charge will also increase in velocity). The time that it subsequently takes for the ion to reach a detector at a known distance is measured. This time will depend on the velocity of the ion, and therefore is a measure of its mass-to-charge ratio. From this ratio and known experimental parameters, one can identify the ion.

In MS/MS fragmentation spectra, once samples are ionized (by ESI, MALDI, EI, etc.) to generate a mixture of ions, precursor ions of a specific mass-to-charge ratio (m/z) are selected (MS1) and then fragmented (MS2) to generate a product ions for detection. The selection-fragmentation-detection sequence can be further extended to the first-generation product ions. For example, selected product ions generated in MS2 can be further fragmented to produce another group of product ions (MS3) and so on. Comparing the TOF MS spectrum of the peptide mixture and three MS/MS fragmentation spectra, we will be able to identify the protein under study.