Peptide sequencing collision-induced dissociation

Peptides inside the mass specttometet ate broken down into smaller units by coUisions with neuttal helium atoms (collision-induced dissociation), and the masses of the individual fragments are determined. Since peptide bonds are much more labile than carbon-carbon bonds, the most abundant fragments will differ from one another by units equivalent to one or two amino acids. Since—with the exception of leucine and isoleucine—the molecular mass of each amino acid is unique, the sequence of the peptide can be reconstructed from the masses of its fragments. 

Sequence information can be obtained for peptides with molecular weights up to 2500 Da. Collision-induced dissociation of larger peptides reveal at least partial sequence information that will often suffice to solve a particular problem. The collision-induced dissociation method has been particularly useful on peptides from proteolytic digests, from which MS/MS data on different peptides can help identify the structure of a digested protein. 

In some cases the identification of proteins based on peptide masses is ambiguous or a suitable match cannot be found. Further information may be obtained by performing a collision-induced dissociation tandem mass spectrometry (CID MS/MS) experiment, in which the peptide molecular ions are fragmented in a collision cell and the resulting product ions are analyzed. These product ions often reveal sequence information that can then be used to identify the protein. 

Yates, J.R., Eng, J.K., Clauser, K.R. and Burlingame, A.L. (1996) Search of sequence databases with uninterpreted high-energy collision-induced dissociation spectra of peptides. J. Am. Soc. Mass Spectrom., 7 (11) 1089-98. 

Figure 2. Workflow of an LC-MS/MS experiment. A mixture of peptides from a protein sample digest is separated by reversed-phase chromatography on a nano-flow HPLC. The peptides elute from the RP column and are ionized by an electrospray source. In the first stage of mass spectrometry, m/z values and charge states for each precursor ion are determined and the most abundant precursor ions are selected for analysis in the second stage. The ions are then fragmented with by collision-induced dissociation (CID) a gas to produce fragment ions which are detected. Using the mass (from MS-1) and sequence information (from MS-2) protein sequence databases are searched to provide peptide identifications and protein matches.

A complementary approach to protein identification is based on peptide seqnencing analysis (PSA) by collision-induced dissociation (CID) in MS-MS. Por proteomics studies, the de novo sequencing of MS-MS spectra is too time-consuming. Therefore, algorithms have been developed to either provide antomated MS-MS spectmm interpretation, or perform protein identification by means of a SEQUEST database search [6-7]. In the latter case, the precursor m/z values are matched against a virtual digestion of all the proteins in the database. Sequence ions are predicted for the peptides that match the precnrsor m/z values, and compared with the measured MS-MS data. A correlation score is calculated for each match. 

There are three main types of mass analyzers in ESTMS-MS instruments triple quadrupole, ion traps, and quadrupole-time-of-flight (Q-TOF). There are several differences between the mass analyzers in MALDI-TOF and in ESI-MS-MS. Unlike in MALDI-TOF-MS, in ESTMS-MS two mass analyzers are used in tandem to increase the sensitivity of the technique. The peptide ions produced by the ESI sources are carried to the first mass analyzer and only peptides of a set miz ratio are selected. The selected ions are then carried to a collision cell where they are subjected to additional fragmentation to produce smaller amino acid ions using a process called as collision induced dissociation (CID). The CID process employs inert gases such as argon for the dissociation of peptides. These smaller amino acid ions are then resolved in the second mass analyzer before sending to the detector. This process essentially enables highly sensitive detection of actual amino acid sequence of the peptides based on the mIz ratios of individual amino acids. 

FIGURE 4.11 ESI-MS-MS spectrum from collision-induced dissociation (CID) of the doubly charged ion m/z 912.3 from 20 pmole/ul of rennin substrate (rat) in 0.1% acetic acid showing the b- and y-ion fragments from the peptide. The amino acid tyrosine (Y) is determined in the sequence using the doubly charged and ions. See Figure 4.2 for the full scan spectrum. 

Much of the success of these so-called soft ionization methods results from the low levels of vibronic excitation imparted to the molecules during the ionization process. Thus molecular ions are less likely to break down and they are mostly preserved intact, even for labile and potentially unstable compounds. Structural information can be obtained by collision-induced dissociation (CID) of these preponderant molecular ions in a tandem mass spectrometer, which can lead directly to the sequence of amino acids in a peptide or the sequence and branching of sugar units in an oligosaccharide. This technique is also referred to as mass spectrometry/mass spectrometry (MS/MS). Today mass spectrometry is capable of determining the amino acid sequence and posttranslational modifications of virtually any protein that can be purified in sulficient quantity. 

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