Sequence coverage

As we cannot obtain any further information from the N-terminal fragments, we should try to supplement our sequence coverage with the information obtained from C-terminal fragments. First the N-terminal amino acid can be identified through a mass difference between the mass of the precursor ion and the heaviest of the y-ion... 

The systematic characterization of secondary protein modifications is different from sequencing a protein. For a systematic analysis of protein modifications, the complete protein sequence should be covered by the investigation. This is difficult to achieve since the enzymatic digestion of a protein is not homogeneous. The sequence coverage depends on the nature of the protein, its quantity, and the enzyme used. Typical... 

To localize the rate of deuterium buildup to specific amides, the analyte protein is fragmented into a collection of peptides using combinations of endo- and exoproteases. Due to the low pH of the quench conditions in which the protein and peptide samples are maintained after deuterium labeling, acid-reactive proteases such as pepsin must be employed. Studies with combinations of acid-reactive en-doproteinases and carboxypeptidases have been employed to achieve greater sequence coverage and higher amide resolution [42, 45]. 

As a general trend, de novo transcriptome assembly studies reveal greater transcriptome complexity than expected and provide a blueprint for further studies. De novo transcriptome assembly of black pepper is a representative example of the application of SOLiD RNA-Seq technology in nonmodel species (29). In that study, 71 million short reads, representing a sequencing coverage per base (depth) of 62X, were used to assemble a total of 22,363 transcripts in root samples. Transcript and functional annotation was performed based on the sequence homology with other species. 

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 P. aeruginosa, despite considerably lower levels of LasR when compared to the E. coli system, the same protocol resulted in detection of a band by in-gel fluorescence, which could be excised and the identification of LasR achieved by proteomic MS methods with 32% total sequence coverage. The methodology was also extended to allow analysis by flow cytometry. In this way, the authors were able to demonstrate that cell density had an effect on LasR labelling. As expected, fluorescence increased with cell density up to the point at which quorum was achieved (around OD600 = 6), beyond which fluorescence began to weaken. 

TOF-TOF instruments can also analyse small intact proteins but this instrument leads to low sequence coverage. Indeed, as observed for CID, proteins above 5000 Da produce sequential b and y fragment ions principally at the termini of the protein. Nevertheless, the most predominant fragment ions correspond to cleavage at the C-terminal to aspartic or glutamic acid residues and at the N-terminal to proline residues in the protein [73],... 

Spot number Identified proteins Accession number Mowse score No. of peptides identified Sequence coverage <%)... 

To test these possibilities, random segments of four to seven residues were inserted into the middle of the EcCM HI helix [95]. The individual libraries (designated L4, L5, L6 and L7) have a maximum theoretical diversity of 160,000 (204), 3.2 x 106 (205), 6.4 x 107 (206), and 1.28 x 109 (207) distinct members, respectively. In each case, transformation of chorismate mutase-deficient bacteria yielded roughly 107 clones, giving fully diverse and redundant coverage of the L4 and L5 libraries, 10 % sequence coverage of the L6 library, and 1 % coverage of the L7 library. 

Gel Band Calculated MW (Da) Measured MW (Da) MS-Fit Search Sequence Coverage (%)... 

Figure 19-26. Sequence and sequence coverage of pVIII (matched sequences are highlighted in boldface). Oxidation of Met ° was detected by peptide mapping. (Reprinted from reference 138, with permission of Elsevier Science B.V.)...

Figure 5. Summary of the strategy applied for a better sequence coverage of the identified proteins in ID gel. The first step is the analysis of the raw peptide mixture by MALDI. The step 2 corresponds to the nanoLC-MS/MS analysis of the peptide digests, and the step 3 corresponds to the off-line fractionation of the peptide digest and MALDI-MS analysis. The combination of these approaches allow a better coverage of the different proteins identified.

The improvement of sequence coverage of the proteins is very important first to enhance the confidence of the different identifications and second to have a better characterization of the proteins, particularly in term of post-translational modifications and/or mutations. 

The same tryptic digest protein sample was analyzed by capillary LC/MS/MS using an ion trap mass spectrometer followed by a database search with SEQUEST. Fig. 4 illustrates the components of an ion trap mass spectrometer. The highly automated data-dependent MS/MS analysis provided excellent sequence coverage for 11 tryptic peptides related to AlAT in a single LC/MS run. A tryptic peptide that corresponds to the AlAT sequence SVLGQLGITK observed at retention time (r,) 30.5 min. was observed in the spectrum. The LC/MS data also provided sequence information on unmatched MALDI peaks. 

The development of the continuous-flow FAB interface by Caprioli et al. [8] was primarily directed at the analysis of peptides (Ch. 4.6). Continuous-flow FAB provided reduced suppression of hydrophilic peptides in peptide mixtures. It was widely applied in the field of peptide characterization and the analysis of proteolytic digests, e.g., in the analysis of a tryptic digest of bovine ribonuclease B before and after treatment with N-glycanase [9]. A single injection of 100 pmol provided ca. 70% sequence coverage. 

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