Tryptic peptide analysis

In their initial experiments, Sangar et al. (28) found foxir primary products in PMDV-infected BHK 21 cells but a re-assessment was made after tryptic peptide analysis of these products had shown that two of them, P56 and PI00, gave similar maps. The second series of experiments (29) showed that, as with other picomavimises, only three primary products 5 - P188, P52, PI 00 - 5 are present. The polypeptide.P56 is apparently part of PI00. 

Figure 9 Tryptic peptide analysis of the YIA antigen and induced P56 polypeptide of FMDV, strain A61. ...

Fig. 12. Tryptic peptide analysis of the induced polypeptide P52 from cells infected with FMDV,...

ABRAHAM, G. and COOPER, P.B. Relations between poliovirus polypeptides as shown by tryptic peptide analysis. J. Gen. 

Fig. 13. Tryptic peptide analysis of the induced polypeptide P88 from cells infected -with FMDV, strains A Bag and a61, ---, Bag ----,...

The soluble peak, B, runs exactly coincident with the large subunit of Fraction I protein (Fig. 4). The identity of this peak with the large subunit has been confirmed by tryptic-peptide analysis, and this is the first definitive identification of a protein that is synthesized by chloroplast ribosomes. It is this author s opinion that chloroplast ribosomes represent a high proportion of the cellular ribosomal complement, not because they make a wide range of different proteins, but because one of their products—the large subunit of Fraction I protein—is a component of one of the most abundant proteins. The reason for the abundance of this protein is its very low efficiency as a catalyst for the ribulose-diphosphate... 

Once the resolution has been optimized as a function of gradient rate, one can continue to fine-tune the separation, raising flow rate and temperature. In a study of temperature and flowrate variation on the separation of the tryptic peptides from rabbit cytochrome c, column performance doubled while analysis time was reduced by almost half using this strategy.97 Commercially available software has been developed to aid in optimization. As a final note, in an industrial laboratory optimization is not completed until a separation has been shown to be rugged. It is a common experience to optimize a separation on one column, only to find that separation fails on a second column of identical type. Reproducibility and rigorous quality control in column manufacture remains a goal to be attained. 

DEAE 25W were operated in tandem to map the peptides generated on tryptic hydrolysis of very large proteins, such as albumin genetic variants.197 A useful review that covers many aspects of peptide analysis is available.198... 

FIGURE 12.5 Human serum tryptic digest analysis. Fractionation in the first LC dimension was performed using a C18 column at pH 10. Fractions were analyzed using NanoEase 0.3 x 150 mm Atlantis d18 column. Approximately 66 lg (400 pmole of semm albumin peptides) was injected on column. Arrow points to a selected albumin peptide illustrating a local column mass overloading. Ten-5mm wide fractions were collected in 1st LC dimension. 

The combination of this top-down proteomics approach, which generates information on the structure of the intact protein, with a bottom-up approach for protein identification (using MS/MS data of tryptic peptides from the collected fractions) has been particularly useful for identifying posttranslational modifications, cotransla-tional processing, and proteolytic modifications in a number of proteins. Examples from our work will be shown to illustrate this hybrid methodology for proteomics analysis. 

All samples were digested with trypsin and analyzed by cIEF in the first dimension followed by LC-MS/MS as described above. Samples were analyzed in duplicate. Sequence searching was performed using OMSSA. Analysis of the soluble fraction yielded a total of 2856 identified proteins, while the insoluble fraction yielded 3227 proteins. Combined, the fresh-frozen sample yielded 3902 protein identifications. The FFPE portion yielded 2845 protein identifications from 14,178 distinct tryptic peptide sequences, on a par with the fresh-frozen soluble fraction. Combining all identifications gave 4145 proteins. While, the soluble fraction and the FFPE extraction yielded similar numbers of protein identification, both found 25% of their respective protein set uniquely (Fig. 20.4). 

The minimum amount of analyte required for analysis depends strongly (as always) on the nature of the substance and on the properties of the ion source. Less than 100 zeptomole of tryptic peptides from bovine semm albumin has been detected by coupling nanoflow LC to an ESI source [264] and 0.4 nM of equine cytochrome c by employing an RF-field focusing funnel to improve transmission [265]. 

The best analysis can be achieved when the complexity of a protein mixture is reduced. This will increase the capacity of the MS instrument for analysis. Considering those with approximately 6,000,000 tryptic peptides, it becomes highly challenging unless mixtures are separated to their components (single proteins or peptides) prior to analysis. 

The most obvious effect of a deficiency in vitamin K in animals is delayed blood clotting, which has been traced to a decrease in the activity of prothrombin and of clotting factors VII, IX, and X (Chapter 12, Fig. 12-17). Prothrombin formed by the liver in the absence of vitamin K lacks the ability to chelate calcium ions essential for the binding of prothrombin to phospholipids and to its activation to thrombin. The structural differences between this abnormal protein and the normal prothrombin have been pinpointed at the N terminus of the 560 residue glycoprotein.e f Tryptic peptides from the N termini differed in electrophoretic mobility. As detailed in Chapter 12, ten residues within the first 33, which were identified as glutamate residues by the sequence analysis on normal prothrombin, are actually y-carboxyglutamate (Gla). The same amino acid is present near the N termini of clotting factors VII, IX, and X. 

Four tryptic peptides containing intact methionine residue were obtained from the tryptic digest of intact nuclease. Amino acid analysis (32) and NH2-terminus determination by the dinitrofluorobenzene method of the BrCN fragments, the tryptic peptides obtained from the BrCN fragments and the four from native nuclease provided the information required to deduce the linear arrangement of the BrCN fragments (25). 

Re-inspection of the DNA sequence analysis data revealed that not only base 19 is C but also base l8. Because both CGT and CGC code for arginine, the corrected sequence now contains an arginine-proline bond, which trypsin splits very slowly. This is probably the reason why both tryptic peptides (positions 1-12 and 7-12) were... 

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