Bottom-up protein sequencing

Accurate mass measurement is important in sequencing, e.g., to distinguish between the isobaric glutamine and lysine, both of which have nominal residue masses of 128 but different exact masses (128.0586 and 128.0950, respectively). Leucine and isoleucine are isomers and cannot be distinguished by CID. However, 

Except When Cleavage Would Occur Before P P 

Cyanogen bromide treatment is a chemical process that cleaves only at methionine residues. 

In the bottom-up strategy the protein is first digested enzymatically. 

LC is used to separate the resulting peptides and introduce them into the mass spectrometer. 

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The strategy for bottom-up protein sequencing using mass spectrometry starts with the precise determination of the molecular mass of that protein using MALDI or ESI. This result allows one to verify the sequence that is determined ultimately and also to judge the homogeneity of the sample. The protein then is subjected to reduction and alkylation of the cysteine bridges. Determination of the molecular mass allows one to determine the number of cysteines present in the protein. 

In the early LC-MS-MS studies, CID is performed with low-energy collisions by means of triple-quadrapole instruments. Under these conditions, series of N-terminal b-ions and C-terminal y-ions result from cleavages at the peptide bond and charge retention on either side, although double-charge tryptic peptide ions tend to favour fragmentation towards more abimdant y-ions. From these ladders of sequence ions in the MS-MS spectrum, the amino-acid sequence of the peptide may be derived. This is the bottom-up protein identification approach (Ch. 18.3.1). 

The high resolving power of FT-ICR-MS can readily be exploited in bottom-up protein identification. A nice example is the identification of high-abundant proteins in a tryptic digest of human plasma without any prior separation. The 2745 peaks in the spectmm could be reduced to 1165 isotopic clusters and 669 unique masses, 82 of which matched tryptic fragments of albumin (93% sequence coverage) and 16 others transferrin (41%) [39]. The same group showed that a theoretically predicted retention time of a tryptic peptide can be applied as an additional protein identification tool, next to its accurate mass acquired in LC-FT-ICR-MS. [40-41]... 

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. 

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... 

In comparison with the bottom-up approach, the top-down approach for de novo protein sequencing is faster and can be applied on proteins in mixture. However, for analysis of post-translational modifications, these two approaches are complementary and a combination of them is interesting. The bottom-up approach allows detection of low-stoichiometric modifications even if a high-stoichiometric modification is missed. Furthermore, enzymatic digestion leads to the loss of all connectivity information from the original protein species. On the other hand, the top-down approach allows observation of the global pattern of... 

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

The protein identification or sequence determination of a protein can be achieved using two different approaches top-down [22, 23] and bottom-up [24], A top-down experiment involves high-resolution measurement of an intact molecular weight and direct fragmentation of protein ions by tandem mass spectrometry (MS/MS) [25], This approach surveys an entire protein sequence with 100% coverage. Post-translational modifications such as glyco-... 

The top-down strategy avoids a disadvantage of the bottom-up strategy in principle, it verifies the complete DNA-predicted sequence of the protein, as both the intact proteins and the sum of the fragments are measured. 

A general outline of the bottom-up mass spectrometry approach for proteome analysis is presented in Figure 3. In general, the mass spectrometry is performed at the peptide level after digesting the protein to obtain the molecular mass and amino acid sequence-specific ions, which are correlated with similar information in the protein or nucleotide database.7 16 Based upon these measurements, the following approaches have evolved. 

The determination of the molecular mass of the peptides generated after proteolysis is called bottom-up or peptide-level spectroscopy. In contrast, the measurements of intact proteins are called top-down or intact protein-level spectrometry. These two methods are not mutually exclusive but are complementary and essential to provide the complete picture of the amino acid sequence and many posttranslational modifications of a protein. These two terms, i.e., bottom-up and top-down, are borrowed from genomics and are analogous to such terms used in the DNA sequencing during genome analysis. 

In databases built using bottom-up approaches, any computational representation believed to be common to all members of a particular domain family can be used. This representation, in conjunction with appropriate searching software, should optimally be able to distinguish all true family members from the background noise of unrelated proteins stored in sequence databases. This is a challenging problem, tackled with varying degrees of sophistication by different approaches. At the most basic level, the representation can consist of a simple pattern of amino acids common to a particular domain. Such an approach is found in... 

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