Tandem mass spectrometry, peptide sequencing molecules

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... 

Sequencing peptides with tandem mass spectrometry was carried out in the early 1980s (Biemann, 1986 Hunt et al., 1986 Hall et al., 1993). Usually the sensitivity and the lengths of sequences achievable were not sufficient to compete with Edman sequencing techniques. In 1988 and 1989, two efficient cold ionization techniques for large molecules were discovered MALDI (Karas and Hillenkamp, 1988) and the electrospray... 

Identification is primarily based on molecular-mass determination, while for an actual structure elucidation LC-MS must be used in combination with tandem mass spectrometry (MS-MS). ESI and APCI are soft-ionization techniques, generating only intact molecule-derived ions, but no fragment ions for most molecules. Therefore, it is frequently applied in combination with MS-MS to achieve more structural information. With respect to qualitative analysis, the use of electrospray LC-MS-MS for peptide sequencing as part of proteomics research is currently an important area. 

Tandem Mass Spectrometry (MS/MS) Random cleavage of a peptide, similar to that from partial hydrolysis with acid, can also be accomplished with mass spectrometry. An intact protein introduced into a mass spectrometer can be cleaved into smaller fragments by collision with gas molecules deliberately leaked into the mass spectrometer vacuum chamber (a technique called collision-induced dissociation, CID). These peptide fragments can be individually selected for analysis using a technique called tandem mass spectrometry (MS/MS). The mass spectra of these random fragments can be compared with mass spectra databases to determine the protein sequence. 

This results in a limited number of peptides which are generally in the range of 500-3000 amu and are thus amenable to analysis by mass spectrometry. Tandem mass spectrometry produces a series of fragments arising from cleavage of the peptide bonds within the molecule and the sequences for each peptide can thus be determined... 

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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