Tryptic peptides sequences

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 uniqueness of the peptide chosen must be taken into account. Metabolites of the protein drug may have the same sequence, or share a common sequence of the product ions being monitored in the MRM-LC/MS method, possibly resulting in interference. Databases are available to search for tryptic peptide sequences of... 

Tryptic peptide sequences are indicated in the figure. lUPAC nomenclature is used for amino acid abbreviation. 

The 1996 ABRF internal sequencing samples consisted of three samples 1) a 28 kD recombinant p-spectrin fi-agment 2) the same P-spectrin fragment with an additional, unique, IS-residue tryptic peptide sequence inserted near its... 

Tryptic peptide sequences are indicated in the figure above the predicted sequence. A lower case a, b, or c following the peptide designation indicates that the peptide was obtained as a mixture of peptides. lUPAC nomenclature is used for amino acid abbreviation. 

Fig. 5.1 Schematic representation of mapping tryptic peptides sequences ( p, p p ) identified by shotgun-proteomics analysis of an unknown (u) strain to database (DB) proteomes of reference strains b, b, b, b ) and analysis of the created matrix of peptide-to-bacterial...

Figure 5.20 LC-MS-MS spectrum of a tryptic peptide derived from spot 13 (see Figure 5.18) of the silver-stained two-dimensional gel of the proteins extracted from the yeast S. cerevisiae, showing the sequence information that may be extracted. From Poutanen, M., Salusjarui, L., Ruohonen, L., Penttila, M. and KaUddnen, N., Rapid Cotn-mun. Mass Spectrom., 15, 1678-1692, Copyright 2001. John Wiley Sons Limited. Reproduced with permission.

Figure 5. Partial peptidic sequences of peptides resulting fi-om tryptic cleavage PME isoforms.

Figure 6.4. Fragmentation spectrum of a tryptic peptide obtained from bovine serum albumin. Peptide sequence LGEYGFQNALIVR, monoisotopic [M + H]+ = 1479.796, monoisotopic [M+2H]2+ =740.402. Upper panel full scan MS spectrum. Lower panel MS/MS spectrum of a doubly-charged ion at 740.7 m/z with a ladder of y ions, the distances between which correspond to amino acid residues (upper row of letters). A shorter series of b ions is also seen (lower row of letters). See Fig. 6.5 for description of nomenclature. Note the often observed phenomenon where multiply-charged ions lose the charge during fragmentation process and, therefore, have higher m/z values than the original parent ion.

Most of the research performed in this field is based on tryptic peptides. As discussed earlier, such peptides contain basic amino acid residues on their C-terminus, which causes formation of the high intensity C-terminal ion series, mostly y-ions. In such peptides the N-terminal ions have lower intensity and do not provide important sequence information. Introduction of a highly basic group, such as dimethylalkyl-ammonium acetyl (DMAA) or tra(2,4,6-trimcthoxyphcnyl)phosphonium acetate into... 

Fig. 11.6. Peptide sequencing by nanoESI-CID-MS/MS from a tryptic digest of bovine serum albumin (BSA) 800 fmol of BSA were used, (a) Eull scan spectrum, (b) fragmentation of the selected doubly charged peptide ion at m/z 740.5. Adapted from Ref. [66] by permission. Nature Publishing Group, 1996.

Konigsberg, W., and R. J. Hill The structure ol human hemoglobins. 111. The sequence of amino acid.s in the tryptic peptides of the a-chain. Journ. Biol. Chem. 237, 2517 -2561 (1962). 

Matsxtbara, H., and E. L. Smith Human heart cytochrome c. Chymo-tryptic peptides, tryptic peptides and the complete amino acid sequence. Journ. Biol. Chem. 238, 2732—2753 (1963). 

The second approach is the tandem mass spectrometric method (Wilm et ah, 1996 Link et ah, 1999 Yates, 2000). This method relies on fragmentation of individual peptides in the tryptic peptide mixture to gain sequence information. Its main advantage is that sequence information derived from several peptides is much more specific for the identification of a protein than a list of peptide masses. The sequence data can be used to search not only protein sequence databases but also nucleotide databases such as expressed sequence tag (EST) databases and, more recently, even... 

Fig. 9. The chymotryptic peptides of al-CB6 Cl, C2, and C3. The tryptic peptides T 1 -T 5 of C2 are indicated. The numbers of residues per peptide are given in parentheses. The amino acid sequences of Cl, C2, and C3 have been described 67,35,36)...

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