Bottom-up sequencing

Peterman, S. M., Dufresne, C. P., and Homing, S. (2005). The use of a hybrid linear trap/FT-ICR mass spectrometer for on-line high resolution/high mass accuracy bottom-up sequencing. J. Biomol. Tech. 16 112-124. 

Strategy followed in bottom-up sequencing proteins using mass spectrometry. 

In bottom-up sequencing, with trypsin as the proteolytic enzyme, it is usually only possible to observe peptides that represent up to 50-70% of the protein thus, only a fraction of the composition is obtained. Furthermore, it is not possible to arrange the peptides in any specific order, so additional digestions with different enzymes are required to generate complementary sets of data. 

MOCUS implemented the Fussell algorithm (Fussell, 1974) for top-down solutions of the fault tree. This algorithm was used in ALLCUT and was modified to be bottom-up in WAMCE T. While cutsets are valuable for qualitative and quantitative purposes, they are not compact. They are thousands of trains (AND sequences) connected to one OR gate. 

Tailoring block copolymers with three or more distinct type of blocks creates more exciting possibilities of exquisite self-assembly. The possible combination of block sequence, composition, and block molecular weight provides an enormous space for the creation of new morphologies. In multiblock copolymer with selective solvents, the dramatic expansion of parameter space poses both experimental and theoretical challenges. However, there has been very limited systematic research on the phase behavior of triblock copolymers and triblock copolymer-containing selective solvents. In the future an important aspect in the fabrication of nanomaterials by bottom-up approach would be to understand, control, and manipulate the self-assembly of phase-segregated system and to know how the selective solvent present affects the phase behavior and structure offered by amphiphilic block copolymers. 

The second and most important finding of this sequencing project was that the difference of 14 Da between the two proteins was not because of a methy-lation of the protein isolated from chicken but because of a substitution of valine for leucine. The MS/MS analysis of the tryptic pieces in which the substitution occurred is shown in Figure 10.11. Had this been a bottom-up... 

In recent years, a novel approach to protein identification emerged, called top-down sequencing. Here the entire nondigested protein is analyzed. Apart from accurate MW measurement, the protein ion is fragmented by the electron capture dissociation (ECD) method (see Chapter 3). This provides in-depth information on the sequence of protein. Such analysis can be performed only with FTICR instruments (see Section 2.2.6) that ensure high resolution and accuracy but, at the same time, they are exceptionally expensive. However, as very large ions are analyzed, even the high accuracy of FTICR is sometimes not sufficient, and it is recommended that such analyses are accompanied by more traditional bottom-up approaches. 

Every one of us working in the held has some bias or other. One of mine concerns the question of macromolecular sequences. The bias is, that the bottom-up approach to the origin of life will never be close to a solution - both conceptually and experimentally - unhl the problem of the onset of macromolecular sequences is clarihed. Obviously the origin of the specihc macromolecular sequences (as opposed to simple polymerizahon) is not an easy queshon to answer, as it is linked to the general problem of structure regulahon. 

The bottle neck of the bottom-up approach is the difficulty of reproducing on paper and/or in the laboratory those processes which have been moulded by contingency - such as the synthesis of specific macromolecular sequences. 

I have stressed in this book that one of the main reasons why the bottom-up approach to the transition to life has not been corroborated experimentally lies, among others, in the fact that the sequence of our macromolecules of life - enzymes, RNA, and DNA - are the products of the vagaries of contingency and by definition it is then impossible to reproduce them in the laboratory. 

I believe that the bottom-up approach to the origin of life will enjoy a considerable boost, when conditions are found for the prebiotic synthesis of many identical copies of long (>30) co-oligopeptide or co-oligonucleotide sequences. This would at least show that the prebiotic synthesis of enzymes and/or RNA is in principle possible. This remains the main problem with the prebiotic RNA world. 

The bottom-up synthesis of metallic nanowires was further applied to construct a nanotransistor device.93 The sequence-specific winding of the homologous nucleic acid carried by the RecA-protein into the duplex DNA was used to address the nucleic acid/protein complex on the DNA scaffold (Fig. 12.27). The subsequent association of the anti-RecA antibody to the protein DNA complex, followed by the association of the biotinylated antiantibody, and the linkage of streptavidin-modified carbon nanotube deposited the tubes in the specific domain of the DNA scaffold. The further... 

A growing number of researchers are focusing on the use of top-down proteomics, a relatively new approach compared to bottom-up, in which structure of proteins is studied through measurement of their intact mass followed by direct ion dissociation in the gas phase. The main advantages over the bottom-up approach are that higher sequence coverage is obtained, it permits... 

---