Introduction
Mass spectrometers consist of three essential parts.
The first, an ionization source, is a device to convert molecules into gas-phase ions.
Two powerful ionization techniques are in common use. The first, matrix-assisted laser desorption ionization (MALDI) creates ions by excitation of molecules isolated from the energy of the laser by an energy absorbing matrix. The laser energy strikes the crystalline matrix to cause rapid excitation of the matrix and the subsequent ejection of matrix and analyte ions into the gas-phase.
The second technique electrospray ionization (ESI) creates ions by application of a potential to a flowing liquid causing the liquid to charge and subsequently spray.The electrospray creates very small droplets of solvent-containing analyte. Solvent is removed as the droplets enter the mass spectrometer by heat or some other form of energy (e.g. energetic collisions with a gas), and multiply-charged ions are formed in the process. The detection limits that can be achieved with ESI have improved with a reduction in the flow rates and hence the use of small diameter columns to achieve separations at low flow rates.
II. Mass Analyzer & Ion Detector
Once ions are created individual mass-to-charge ratios (m/z) are separated by a second device, a mass analyzer, and transferred to the third device, an ion detector. A mass analyzer uses some physical property, e.g. electric or magnetic fields or time-of-flight, to separate ions of a particular m/z value that then strike the ion detector. The magnitude of the current produced at the detector as a function of time (e.g. the physical field in the mass analyzer is changed as a function of time or the time it takes the ion to move a certain distance) is used to determine the m/z value of the ion. This figure shows an ion trap mass spectrometer with ions trapped in the electric field.By changing the characteristics of the field ions can be manipulated and ejected from the trap to a detector. The time component as a function of the field is what determines the m/z value of the ions.
III. Tandem mass spectrometers
As mentioned before, a tandem mass spectrometer is very useful for gaining structural information about molecules . In the first stage a collection of ions is created in the ion source of the mass spectrometer. The ions are allowed to pass through the first mass analyzer and collision cell and their m/z values are measured in the second mass analyzer. Based on the data collected in the initial measurement, the first mass spectrometer is set to pass just one m/z value. This ion enters the collision cell and collides with argon. The kinetic energy of ions is converted to vibrational energy and the ions fragment. The m/z values of fragment ions are then determined in the second mass spectrometer.
Many types of tandem mass spectrometers have been developed and new innovations in tandem mass spectrometers allow greater automation and efficiency in data acquisition. Data can be generated in a data-dependent manner through interaction of the m/z data in each scan with a computer program to control the type of experiment performed. For example, a scan of the mass range can reveal the presence of several ions above a preset ion-abundance threshold. The computer can signal to the instrument to perform tandem mass spectrometry on each of the ions, thus improving the efficiency of data acquisition, particularly during separations when ions appear for only a brief period of time.
IV. Determining the amino acid sequence of a peptide.
By using tandem mass spectrometry, data specific to an individual peptide is collected. Fragmentation information can be used to determine the amino acid sequence of a peptide.
Shown is the manner in which peptides fragment by the bonds that have been observed to dissociate. The most common fragment ions are the b and y-type ions, which provide overlapping information about the sequence.
b ions <-------------------------------------> y ions
By calculating the molecular weight difference between ions of the same type the sequence can be determined.
The SEQUEST software, developed in the Yates laboratory, uses the fragmentation information of a tandem mass spectrum to search through the complete protein database of Sacchromyces cerevisiae to identify the sequence which best fits the fragmentation pattern.
SEQUEST
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Performs sequencing and identification by matching unknown MS/MS spectra to sequence in a database |
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Finds all peptides that matches the input masses. |
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Calculates a preliminary score based upon matching ion intensities of predicted frament ions to peaks in the experimental spectrum. |
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Calculates final scores by performing cross correlations of theoretical spectra of the top N preliminary scoring peptides against the input spectrum. |
Expected fragmentation patterns can be predicated from sequence and then compared to the spectrum.
In these figures the expected b-ions (in red) and y-ions (in blue) are compared to the acquired spectrum.
In the process of finding the sequence, that best fits the spectrum, the protein from which this sequence is derived is identified. Here is an example of the output.
V. Conclusion
An advantage of this approach is that each peptide tandem mass spectrum represents a unique piece of information, consequently matching one or more tandem mass spectra to sequences in the same protein provides a high level of confidence in the identification and enables the identification of proteins present in mixtures. This process has been automated in the software SEQUEST.
Definitions:
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Mass-to-charge ratio (m/z): Mass spectrometers measure the mass to charge ratios of ions. In MALDI and electrospray ionization, peptides are typically ionized by the addition of one or more protons. Thus, a peptide of molecular weight 1000 daltons will have a m/z value of 1001 after ionization by the addition of one proton and 501 with the addition of two (M+2H)+2. |
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Collision-induced dissociation (CID): One method of energetically activating ions to dissociate. Typically, a gas-phase collision cell filled with argon gas is used to subject ions to low energy collision (10-50 eV) causing energetic excitation. As ions become energetically excited, covalent bonds dissociate to produce structurally informative fragment ions. Often the molecular structure of the ion can be postulated from the fragmentation pattern, or in the case of peptides, the amino acid sequence deduced. |
References (PDF)
Functional genomics by mass spectrometry
Mass spectrometry and proteomics
Advances in mass spectrometry for proteome analysis
Trends in automation and mass spectrometry for proteomics
Using mass spectrometry for quantitative proteomics
Protein complexes and analysis of their assembly by mass spectrometry
Mass spectrometry - from genomics to proteomics
Trends in automation and mass spectrometry for proteomics
Measuring gene expression by quantitative proteome analysis