Peptides digestion and

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.

Another subclass of proteases attacks internal peptide bonds and Hberates large peptide fragments. Bromelain, a plant protease derived from the stem of the pineapple plant, can even produce detectable semm proteolysis after oral adrninistration (180). Oral therapy with bromelain significantly reduces bmising that stems from obstetrical manipulations (181). Bromelain—pancreatin combinations have been more effective in digestive insufficiency compared to either pancreatin or placebo (182,183). Bromelain may also enhance the activity of antibiotics, especially tetracycline, when adrninistered concurrently (184). 

Tandem mass spectrometry (MS/MS) is another common approach used for protein identification. In this method, proteins are digested and the resulting peptides are ionized directly from the liquid phase by... 

M Yoshioka, RH Erickson, YS Kim. Digestion and assimilation of proline-con-taining peptide by rat intestinal brush border membrane carboxypeptidases. Role... 

Proteomics ultimately hinges upon protein identification to reveal the meaning behind the masses, spots, or peaks detected by other means. Because fraction collection is a natural component of HPLC separations, intact proteins can be readily collected either for direct analysis or for proteolytic digestion and identification using peptide mass fingerprinting (PMF) in conjunction with matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). 

The ability to resolve and characterize complicated protein mixtures by the combination of 2DLC and online mass spectrometry permits the combination of sample fractionation/simplification, top-down protein mass information, and bottom-up peptide level studies. In our lab, the simplified fractions generated by 2D(IEX-RP)LC are digested and analyzed using common peptide-level analysis approaches, including peptide mass fingerprinting (Henzel et al., 1993 Mann et al., 1993), matrix-assisted laser desorption/ionization (MALDI) QTOF MS/MS (Millea et al., 2006), and various capillary LC/MS/MS methodologies (e.g., Ducret et al., 1998). 

J. Gao, J.D. Xu, L.E. Locascio, and C.S. Lee, Integrated microfluidic system enabling protein digestion, peptide separation, and protein identification. Anal. Chem. 73, 2648-2655 (2001). 

The rapid development and sensitivity of the mass spectrometric methods can be foreseen and in the near future the labeling can be more frequently eliminated. The identification of the cross-linked peptide can be detected first with immunological methods and then the digested and cleaved fragments with specific tandem MS techniques. The different photophores hold discrete MS fingerprints, which allow fast recognition of the modified sites. 

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