MALDI ladder sequencing

In the ladder sequencing method, the oligonucleotide is enzymatically digested to subsequently remove either 3 - or 5 -terminal residues. The sequence can be derived from the mass change after each cleavage. Ladder sequencing was demonstrated using either MALDI-MS or ESI-MS [27]. 

Sequence information can also be obtained using Edman degradation to remove amino-terminal residues from a peptide, to produce a collection of peptides. MALDI-TOF can then be used to obtain the peptides masses, and the sequence determined by mass difference between consecutive peptides. This methodology is called protein ladder sequencing, and allows information to be obtained for up to 30 residues. This method is useful for the identification of posttranslational modifications, such as phosphorylated amino acid residues.24... 

The following ion-activation techniques have been used at one time or other to sequence peptides (1) fast atom bombardment (FAB) ionization, (2) CID—tandem MS (MS/MS), (3) ESI in-source CID, (4) MALDI ion-source decay, (5) MALDI postsource decay (PSD), (6) electron-capture dissociation (ECD) and electron-transfer dissociation, and (7) peptide ladder sequencing. Because of the lack of space, only (2) and (4) will be discussed further. 

Oligosaccharides can also be incorporated into matrix crystals and gasified. Actually, you should even be able to sequence the oligosaccharides with the MALDI-TOF (ladder sequencing see Section 7.6.5 and Stahl et al. 1994). In principle, MALDI brings any polymer into the gas phase, even polyethylene glycol. However, you have to supply such neutral polymers with a positive charge. Otherwise, the electric field cannot accelerate them into the TOE These polymers are therefore complexed with metal ions (whose MW, of course, needs to be taken into account). 

The MW determination by means of MALDI-TOF is so exact that the mass difference between a peptide with n amino acids and the same peptide minus one amino acid allows the identification of the missing amino acid. Even Asp (MW 115.1) and Asn (MW 114.1) can be determined this way. High-end devices even detect whether the peptide is lacking Lys (MW 146.190) or Gin (MW 146.146). However, the peptides should not be too large (maximum 30 amino acid residues). Following Chait et al. (1993), ladder sequencing is based on the accuracy of MALDI-TOF and a modified Edman reaction (Figure 7.10). 

Once you have the peptide ladder, you get the sequence of your peptide within minutes from a MALDI-TOF run. Because you can produce ladders from different peptides (in different tubes) at the same time, sequencing with peptide ladders and MALDI-TOF is faster than with the traditional Edman method Table 7.4. In addition, you need less peptide. This is true only, however, if it works, because as any other method ladder sequencing also has its problems. 

Example 8.4 The peptide of Example 8.3 (i.e., Thr-Gly-Met-Leu-Phe) is sequenced by the ladder sequencing technique. At what miz values will ions be observed in the MALDI mass spectrum after digestion with carboxypeptidase Y ... 

Peptide Ladder Sequencing The treatment of a glycopeptide with a carboxypeptidase or aminopeptidase to generate peptide ladders, followed by MALDI-MS analysis of those ladders, is another feasible approach to identifying the site of glycosylation. This approach may not be effective with multiglycosylation sites because the enzymatic activity of these proteases is impaired at or near the site of glycosylation [84]. 

The ability of FAB mass spectra to deliver peptide sequence information was soon recognized [15,130]. Initially, the sequence was derived from fragment ions observed in the full scan spectra [15,96]. Another approach to sequence information is to subject the peptide to enzymatic hydrolysis by a mixture of several carboxy-peptidases to produce a series of truncated molecules. The FAB spectrum of the mixture then reveals the C-terminal sequence [131,132]. In the MALDI community, this approach became known as peptide ladder sequencing [133]. 

Figure 18.5. MALDI-TOF mass spectrum from protein ladder sequencing of the N-terminal peptide of cofilin (G-S-R-S-G-V-A-...). A stepwise degradation was carried out in the presence of 5% phenylisocyanate/95% phenylisothiocyanate. The peptide was subjected to two cycles of ladder generating chemistry. ...

This approach to analyze exonuclease ladders seems to be a particularly promising tool to determine rapidly the sequence of oligonucleotides. Compared to conventional methods, based on the laboratory-scale preparation of a large number of samples at different enzyme and substrate concentrations followed by MALDI-MS analysis, the microfluidics approach offers the advantage of saving time and material. Furthermore, due to the limited sample handling, the risks encountered when manipulating biomolecules are also reduced as well as that of sample contamination. 

The MALDI-technique is not limited to proteins/peptides. With a suitable matrix you can also bring DNA and RNA into the gas phase. What for For example, you can sequence oligos by means of oligonucleotide ladders (Limbach et al. 1995). The smallest MW difference between DNA bases—between adenine and thymine—amounts to at least 9 Dalton. For RNA bases, the smallest difference—between cytosine and uracil—lies at 1 Dalton. 

The most popular method is to completely digest the proteins after the blot. For this, you identify your protein on the (unblocked ) blot by means of protein stain, cut out the blot piece, and add a selective protease. This creates peptides that are separated by HPLC. You can sequence these peptides following Edman (see Section 7.6.4) or by means of a peptide ladder and MALDI-TOF (see Section 7.6.6). 

The cycle can be repeated at will. The result is a ladder of PIC peptides with n, n-1, n-2, n-3 amino acids and a residue peptide with a free N-terminus. This one also becomes blocked with PIC after the last cycle. Now, the MALDI-TOF separates the PIC peptide ladder and measures the MW of the individual PIC peptides. By their MW differences, you can identify the cut-off amino acids. Then, the peptide sequence can be read directly from the spectrum (Figure 7.10). 

Time 1 day 1 day for 10 cycles. The MALDI-TOF run takes only minutes. If you produce ladders for several samples at the same time, you can sequence several samples in a day. 

Various forms of tandem mass spectroscopy (MS/MS) have also been used in the analysis of biomolecules. Such instruments consist of an ionisation source (ESI or MALDI or other) attached to a first mass analyser followed by a gas-phase collision cell. This collison cell further fragments the selected ions and feeds these ions to a second mass detector. The final mass spectrum represents a ladder of fragment ions. In the case of peptides the collision cell usually cleaves the peptides at the amide bond. The ladder of resulting peptides reveals the sequence directly [496]. Thus, tandem MS instruments, such as the triple quadrupole and ion-trap instruments have been routinely applied in LC-MS/MS or ESI-MS/MS for peptide sequencing and protein identification via database searching. New configurations, which have been moving into this area include the hybrid Q-TOF [498], the MALDI-TOF-TOF [499] and the Fourier transform ion cyclotron resonance instruments [500]. 

MALDI-TOF-MS analysis of the ladders generated, the spectrum shown in Figure 8.24 was obtained. Determine the sequence of the protein. 

The oligonucleotide 5 -snake venom phosphodiesterase. At what m/z values will the 5 -sequence ladders be seen in the negative-ion MALDI mass spectrum ... 

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