Peptide mass spectrometry fragmentation

See also Chemical Structure Information from Mass Spectrometry Chromatography-MS, Methods Fast Atom Bombardment Ionization in Mass Spectrometry Fragmentation In Mass Spectrometry Hyphenated Techniques, Applications of in Mass Spectrometry Medical Applications of Mass Spectrometry MS-MS and MS Nucieic Acids and Nucieotides Studied Using Mass Spectrometry Peptides and Proteins Studied Using Mass Spectrometry Quadrupoies, Use of in Mass Spectrometry Sector Mass Spectrometers Surface induced Dissociation in Mass Spectrometry Time of Fiight Mass Spectrometers. 

See also Biochemical Applications of Mass MS-MS and MS Organometallics Studied Using Spectrometry Fragmentation in Mass Spectrometry Mass Spectrometry Peptides and Proteins Studied Ion Energetics in Mass Spectrometry Ionization The- Using Mass Spectrometry Plasma Desorption loniza-ory IR Spectroscopy Sample Preparation Methods tion in Mass Spectrometry Spectroscopy of Ions. 

Tandem mass spectrometry (MS/MS) produces precise structural or sequence information by selective and specific induced fragmentation on samples up to several thousand Daltons. For samples of greater molecular mass than this, an enzyme digest will usually produce several peptides of molecular mass suitable for sequencing by mass spectrometry. The smaller sequences can be used to deduce the sequence of the whole protein. 

Mass spectral fragmentation patterns of alkyl and phenyl hydantoins have been investigated by means of labeling techniques (28—30), and similar studies have also been carried out for thiohydantoins (31,32). In all cases, breakdown of the hydantoin ring occurs by a-ftssion at C-4 with concomitant loss of carbon monoxide and an isocyanate molecule. In the case of aryl derivatives, the ease of formation of Ar—NCO is related to the electronic properties of the aryl ring substituents (33). Mass spectrometry has been used for identification of the phenylthiohydantoin derivatives formed from amino acids during peptide sequence determination by the Edman method (34). 

Tandem mass spectrometry (MS/MS) is a method for obtaining sequence and structural information by measurement of the mass-to-charge ratios of ionized molecules before and after dissociation reactions within a mass spectrometer which consists essentially of two mass spectrometers in tandem. In the first step, precursor ions are selected for further fragmentation by energy impact and interaction with a collision gas. The generated product ions can be analyzed by a second scan step. MS/MS measurements of peptides can be performed using electrospray or matrix-assisted laser desorption/ionization in combination with triple quadruple, ion trap, quadrupole-TOF (time-of-flight), TOF-TOF or ion cyclotron resonance MS. Tandem... 

Table 5.5 Nomenclature of the ions formed in the mass spectral fragmentation of polypeptides. From Chapman, J. R. (Ed.), Protein and Peptide Analysis by Mass Spectrometry, Methods in Molecular Biology, Vol. 61, 1996. Reproduced by permission of Humana Press, Inc. 

Like peptide oligomers, peptoids can be analyzed by HPLC and by mass spectrometry. They can be sequenced by Fdman degradation [13] or by tandem mass spectrometry [14] since, like polypeptides, they conveniently fragment along the main chain amides [15, 16]. 

Complex peptide mixmres can now be analyzed without prior purification by tandem mass spectrometry, which employs the equivalent of two mass spectrometers linked in series. The first spectrometer separates individual peptides based upon their differences in mass. By adjusting the field strength of the first magnet, a single peptide can be directed into the second mass spectrometer, where fragments are generated and their masses determined. As the sensitivity and versatility of mass spectrometry continue to increase, it is displacing Edman sequencers for the direct analysis of protein primary strucmre. 

Figure 2.4. Peptide fingerprinting by MALDI-TOF mass Spectrometry. Proteins are extracted and separated on by 2D gel electrophoresis. A spot of interest is excised from the gel, digested with trypsin, and ionized by MALDI. The precise mass of proteolytic fragments is determined by time-of- flight mass spectrometry. The identity of the protein is determined by comparing the peptide masses with a list of peptide masses generated by a simulated digestion of all of the open reading frames of the organism.

Figure 2.5. Tandem mass spectrometry. A. A peptide mixture is electrosprayed into the mass spectrometer. Individual peptides from the mixture are isolated (circled peptide) and fragmented. B. The fragments from the peptide are mass analyzed to obtain sequence information. The fragments obtained are derived from the N or C terminus of the peptide and are designated "b" or "y" ions, respectively. The spectrum shown indicates peptides that differ in size by the amino acids shown.

One attempt to overcome these disadvantages has been to use multidimensional liquid chromatography (LC) followed directly by tandem mass spectrometry to separate, fragment and identify proteins (Link et al., 1999). In this process, a denatured and reduced protein mixture is digested with a protease to create a collection of peptides (Fig. 2.6). The peptide mixture is applied to a cation exchange column and a fraction of these peptides are eluted based on charge onto a reverse-phase column. The... 

MS/MS Duty Cycle Typical MS/MS analysis is a serial process, relying on the selection of precursors (peptides) in MS mode, followed by high-energy fragmentation in MS/MS. This process is termed data dependent acquisition (DDA). The duty cycle for the completion of MS and MS/MS cycles (the time necessary for MS/MS spectrum acquisition) is of primary importance. When the separation performance is viewed from the mass spectrometry perspective, the peak capacity can be characterized by the number of MS/MS scans, yielding successful... 

No tandem MS experiment can be successful if the precursor ions fail to fragment (at the right time and place). The ion activation step is crucial to the experiment and ultimately defines what types of products result. Hence, the ion activation method that is appropriate for a specific application depends on the MS instrument configuration as well as on the analyzed compounds and the structural information that is wanted. Various, more or less complementary, ion activation methods have been developed during the history of tandem MS. Below we give brief descriptions of several of these approaches. A more detailed description of peptide fragmentation mles and nomenclature is provided in Chapter 2. An excellent review of ion activation methods for tandem mass spectrometry is written by Sleno and Volmer, see Reference 12, and for a more detailed review on slow heating methods in tandem MS, see Reference 13. 

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