Peptide digest fragments

Due to the structural complexity of proteins, proper confirmation of product identity generally requires the use of several different techniques in parallel. For example, use of RP-HPLC and capillary electrophoresis in parallel provides a powerful means for proving identity of a particular peptide fragment since these two techniques exhibit independent elution profiles for typical peptide digests (1) with RP-HPLC separating on the basis of hydrophob-icity and capillary electrophoresis separating on the basis of charge. 

The combination of MALDI-TOF MS and capillary LC/MS/MS was recently described for the identification of disease state markers in human urine. In this study, urine proteins obtained from emphysema patients were separated on 2-D gels and selected spots were digested with trypsin and analyzed by MALDI-TOF. A database search using Protein Prospector identified a potential biomarker for emphysema as human alpha-1-antitrypsin (AlAT). The corresponding MALDI spectrum contained nine out of 18 peptides with masses that match the expected tryptic digest fragments for AlAT. 

A tryptic fragment of connectin, the 400-kDa peptide, has been demonstrated to cause aggregation of myosin filaments. Further digested fragments of chain weights less than 40 kDa lost this ability (Kimura et al., 1984b). 

The Sequazyme C-peptide sequencing kit enables peptide digestion followed by analysis of sequentially truncated peptides using MALDI-TOF-MS. Before digesting and analyzing unknown samples of the peptide (adrenocorticotropic hormone), the activity of the enzyme carboxypeptidase Y (CPY) was checked by dilution experiments with ammonium citrate buffer (pH = 6.1), CPY dilution and the peptide standard angiotensin I. The adrenocorticotropin ACTH (18-39) fragment (0.3 pL) was transferred as a solution of 5 x 10 5M in a 1 1... 

Figure 5. Mapping of protein polymorphisms by mass spectrometry. A Major Urinary Protein from Mus spretus was purified and Lys-C digestion fragments were mass measured by MALDl-ToF mass spectrometry. Four of the peptides had the same masses as the equivalent protein from Mus domesticus. However, for two of the remainder, of novel mass, tandem mass spectrometry led to the identification of an amino acid change that was consistent with the new peptide mass. (J is used to define leucine or isoleucine, which cannot be distinguished by this type of mass spectrometry).

Additional techniques can provide further information when the structure of a peptide is unknown. One method is to carry out the protein digestion in water that is labeled with 50% 0, yielding an isotopically labeled ( 0/ 0) doublet for every cleaved peptide. When such peptides are fragmented the y ions (and other C-terminal ions) can be readily identified, as each will retain the doublet, whereas the N-terminal fragments will have normal isotope distributions that contain only the isotope. 

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