Mass spectrometry : Structural Information

Structural information of proteins can be determined by digesting proteins with specific endoprotease like trypsin, AspN, and GluC. MALDI is one of the best spectrometric techniques for direct analysis of peptide mixtures. Signals of peptides are suppressed because there is a competition for charge or optimal position in the matrix. Therefore signal intensity does not necessarily reflect the quantities of different peptides in the mixtures. Complete sequences can be obtained from a combination of spectra recorded in different modes, like positive and negative, matrices, and different enzyme digestion.

Sequence information is also possible to get from PSD. This is possible by controlling the voltage of the reflector, which results in different m/z ranges on the detector and generates a PSD spectrum. A large sample amount is required since only a small fraction goes under PSD. Additionally, the fragmentation can not be controlled since different site of a peptide can get fragmented. This makes it very had to get complete sequences of a peptide. Alternatively, collision cells are included to the flight tube in MALDI-TOF by some manufacturers, to have controlled fragmentation by collision-induced dissociation.

Large amounts of in-source fragmentation occurs before initiation of the acceleration voltage called in-source decay in delayed extraction equipped MALDI-TOF, which only yield long regions of sequence-specific ions (6). C-terminal sequence ladders can be generated by digestion of peptides with carboxypepsidase and N-terminal sequence ladders can be obtained by Edman degradation using low percentage of phenyltiocarbamate rather than phenyoisothiocarbamate in the coupling reaction. These ladders in the mixtures of peptides can be an alternative to sequence-specific fragment ions. This process often gives a lot of sequence information.

Secondary protein modifications can also be determined using MALDI-TOF-MS. The steps involved in determining secondary modifications are measuring mass of the intact protein, knowing the protein’s primary sequence, and generating site-specific information by direct mass spectrometric peptide mapping of a mixture derived by proteolytic cleavage of the proteins. In tandem (TOF/TOF) configurations, MALDI instruments can provide protein sequence data, as well.

In tandem mass spectrometry, an ion of a particular mass is selected (that's the first stage of the analysis) and fragmented. Its constituent fragment ions are then mass-analyzed a second time (that's the tandem stage) to reveal data about the molecule's structure or sequence; single-stage TOF instruments lack this capability (though some fragmentation does occur via "post-source decay" as the ions traverse the flight tube).

Some companies offer tandem MALDI instruments based on hybrid mass analyzer configurations. Applied Biosystems' QSTAR, for instance, couples an optional MALDI source with a quadrupole-time-of-flight mass analyzer, as does Waters Corporation's MALDI Q-Tof Premier.