Tandem mass spectrometry, peptide sequencing

L. Ngoka and M. L. Gross, Multistep tandem mass spectrometry for sequencing cyclic peptides in an ion-trap mass spectrometer, J. Am. Soc. Mass Spectrom. 

However, interpretation of, or even obtaining, the mass spectrum of a peptide can be difficult, and many techniques have been introduced to overcome such difficulties. These techniques include modifying the side chains in the peptide and protecting the N- and C-terminals by special groups. Despite many advances made by these approaches, it is not always easy to read the sequence from the mass spectrum because some amide bond cleavages are less easy than others and give little information. To overcome this problem, tandem mass spectrometry has been applied to this dry approach to peptide sequencing with considerable success. Further, electrospray ionization has been used to determine the molecular masses of proteins and peptides with unprecedented accuracy. 

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. 

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... 

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. 

The major advantage of the tandem mass spectrometry approach compared to MALDI peptide fingerprinting, is that the sequence information obtained from the peptides is more specific for the identification of a protein than simply determining the mass of the peptides. This permits a search of expressed sequence tag nucleotide databases to discover new human genes based upon identification of the protein. This is a useful approach because, by definition, the genes identified actually express a protein. 

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.

Figure 2.6. LC-tandem mass spectrometry to examine complex mixtures. The mixture of many different proteins is digested to yield peptides and the peptides are resolved into fractions hy cation exchange chromatography followed by reverse phase chromatography. The fractionation steps resolve the peptides into fractions that he processed hy tandem mass spectrometry to yield sequence information suitable for database searching.

Tandem mass spectrometry has become an important tool for determining the sequence of amino acids in protonated peptides98 and the sequence of bases in deprotonated nucleic acids such as DNA.99 Despite the importance and widespread use of CID-MS to sequence peptides and nucleic acids, the mechanistic details of the dissociation processes are poorly understood. A better understanding of the... 

J. A. Taylor and R. S. Johnson. Sequence Database Searches via de novo Peptide Sequencing by Tandem Mass Spectrometry. Rapid Commun. Mass Spectrom., 11, no. 9 (1997) 1067-1075. 

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... 

Even though the MALDI peptide mass mapping technique is very powerful, it has limitations. It requires well-separated proteins, is less sensitive than identifications based on electrospray tandem mass spectrometry, can only identify proteins whose complete sequences are available in databases, and does not produce redundant information. 

Fig. 7. Protein identification with electrospray tandem mass spectrometry and a triple quadrupole mass spectrometer. Fragment spectra of several peptides are generated during one investigation. From the fragment spectra short sequence stretches can be read. Together with their mass location in the peptide of the measured mass, they can be used to specifically identify a protein in the database. Because the protein identification depends only on one peptide, several proteins can be identified from one sample.

Continuous ion series are often generated when multiply charged peptide ions are fragmented. The problem in de novo sequencing with electrospray tandem mass spectrometry lies in minimizing the error rate of the interpretation. There are two different approaches to this problem ... 

Johnson R.S. and Taylor J.A. (2000), Searching sequence databases via de novo peptide sequencing by tandem mass spectrometry, in Methods in Molecular Biology, Vol. 146, Mass Spectrometry of Proteins and Peptides, pp. 41-61, Chapman J.R., Ed., Humana Press, Totowa, NJ. 

Peptide and protein sequencing and its importance in the proteomics field were discussed in Section 2.2.3. The following gives a brief description of the mass spectrometry methods used to achieve sequencing. First, to produce protein or oligonucleotide structural/sequence information by mass spectro-metric techniques, one needs to use tandem mass spectrometry (MS-MS). In this technique, a sample is first fragmented and analyzed in one mass spec-... 

Coon JJ, Syka JEP, Shabanowitz J, Hunt DF. Tandem mass spectrometry for peptide and protein sequence analysis. Biotechniques 38, 519-523, 2005. 

Peptide sequencing via post-source decay and tandem mass spectrometry. 339... 

Taylor, J. A., and Johnson, R. S. (2001). Implementation and uses of automated de novo peptide sequencing by tandem mass spectrometry. Anal. Chem. 73 2594-2604. 

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