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In order to obtain peptide sequence information by mass spectrometry, fragments of an ion must be produced that reflect structural features of the original compound. Fortunately, most peptides are linear molecules, which allow for relatively straightforward interpretation of the fragmentation data. This is accomplished by colliding the ions with an inert gas. The fragments then monitored via mass analysis.

Tandem mass spectrometry allows for a heterogeneous solution of peptides to be analyzed by filtering the ion of interest into the collision cell, structural information can be derived on each peptide from a complex mixture. The fragment ions produced in this process can be separated into two classes. Once class retains the charge on the N-terminus and fragmentation occurs at a, b, and c. The second class of fragment ions retain the charge on the C-terminus and fragmentation occurs at x, y, and z. Most fragments are obtained from cleavage between a carbonyl and amide bond.

When PMF fails, fragments in the CID spectrum can provide crucial information. The data can be used in two ways:

• Uninterpreted fragment ion masses can be used in correlative database searching to identify proteins whose peptides would likely yield similar CID spectra under the same fragmentation conditions. Probability-based matching is used here.
• Peaks of the mass spectrum can be interpreted, either manually or automatically, to derive partial de novo peptide sequences that can be used as standard database queries.

PMF is an analytical technique for protein identification using data from intact peptide masses. A protease such as Trypsin is used to cleave a protein of interest. The masses of the proteins are measured with a mass spectrometer. Each protein can be uniquely identified by the masses of its constituent peptides since protein masses are extremely discriminatory. The Accuracy of PMF depends on quality and relative intensities of the peaks, mass accuracy of the instrument, and interfering factors such are PTMs. PMF can only be used to identify proteins which are sequenced. Therefore PMF is best suited to those organisms whose cDNA protein sequence data is available in a database. It must be noted that even small differences in mass can result in faulty results since PMF accuracy depends entirely on the accurate correlation of determined and predicted masses.

Once separated by mass analyzer, ions reach ion detector which generates a current signal from incident ions. The most commonly used detector is the electron multiplier.3 different types of detectors: Electron multipliers, dynolyte photomultiplier, microchannel plates.

Electron multiplier

A conversion dynode is used to convert either negative or positive ions into electrons. These electrons are amplified by a cascade effect in a horn shape device, to produce a current. This device, also called channeltron, is widely used in quadrupole and ion trap instruments.

Dynolyte photomultiplier

A hybrid mass analyzer is a mixture of two or more mass analyzers. If done correctly, a hybrid can couple the benefits of different mass analyzers.

qTOF

qTOF combines the stability of a quadrupole analyzer with the high efficiency, sensitivity, and accuracy of a TOF reflectron mass analyzer. The quadrupole can act as any simple quadrupole analyzer to scan across a specified m/z range. However, it can also be used to selectively isolate a precursor ion and direct that ion into the collision cell. The resultant fragment ions are then analyzed by the TOF reflectron mass analyzer.

qTOF exploits the quadrupole’s ability to select a particular ion and the ability of TOF to achieve simultaneous and accurate measurements of ions across full mass range. qTOF offers significantly higher sensitivity and accuracy over tandem quadrupole instruments when acquiring full fragment mass spectra.

FT-ICR mass analyzer is the most complex and difficult to operate. It offers the highest resolution, mass accuracy, and sensitivity.

FTMS is based on the principle of monitoring a charged particle’s orbiting motion in a magnetic field. While ions are orbiting, a pulsed RF signal is used to excite them. This allows the ions to produce detectable image current by bringing them into coherent motion and enlarging the radius of the orbit. The image current can generated by the ions can then be Fourier-transformed to obtain component frequencies of different ions, which correspond to their m/z. All ions with the same m/z value will orbit with the same cyclotron frequency in a uniform magnetic field. Since frequencies can be obtained at high accuracy, m/z can also be determined at high accuracy. In addition to high resolution, FTMS offers the ability to perform MSn. It is capable of ejecting all but the ions of interest.

Both ESI and MALDI can be used with ion trap analyzers. Ion trap consists of a chamber surrounded by a ring electrode and two end-cap electrodes. It can trap ions in a radio frequency quadrupole field.

Ions above a certain m/z threshold remain in the trap. Ions are ejected based on applied voltage. So a mass spectrum can be obtained by gradually increasing the voltage. Alternatively, an inert gas can be inserted to fragment the ions. Multiple rounds of fragmentation can be used.

Ions trap is capable of isolating ion species by ejecting all other from the trap. This is usually done to repeatedly fragment ions of interest. This significantly increases the amount of structural information which can be gathered.

TOF - Time of flight mass analyzers are the simplest mass analyzers. TOF analysis is based on accelerating a group of ions to a detector where all of the ions are given the same amount of energy through an accelerating potential. Given the same push, lighter ions reach the detector before the heavier ones. Mass, charge, and kinetic energy of the ions affect the arrival time and the detector.

Unlike quadrupole instruments, electric field is not required to separate ions. MALDI-TOF/TOF or hybrid analyzers are extremely sensitive. TOF separates ions in space.

Reflectron

The quadrupole is the most widely used analyzer due to its ease of use, mass range covered, good linearity for quantitative work, resolution and quality of mass spectra. Reasonably priced.

The main characteristics are:

• Working mass range: 10 to 4000 A.M.U.
  
• Resolution: usually operated at a resolution = 1000, but resolution can be reasonably pushed up to 4000

• Mass accuracy: 0.1 to 0.2 A.M.U.
• Scan speed: up to 5000 A.M.U per second
  

A quadrupole can be operated in RF-only mode, which allows ions of any m/z ratio to pass through, or in scanning mode, where a potential difference is applied and the instrument acts as a mass filter.

Analytical instruments in general have variations in their capabilities as a result of their individual design and intended purpose. Mass analyzers also have their variations their strengths and weaknesses associated with each variation. A mass analyzer measures gas phase ions with respect to their (m/z). It is important to remember that mass analyzers measure m/z ratio, not mass. Quadrupoles and TOFs separate ions in space. Ion trap separates ions in time.