Mass spectroscopy peptides

Methods to measure the structure of biopharmaceuticals include tryptic peptide mapping, HPLC, capillary electrophoresis (CE), mass spectroscopy, and circular dichroism spectra. 

Although a number of assays and technologies are available to characterize and test protein molecules, such as peptide mapping, protein sequencing, carbohydrate analysis, electrophoresis, ELISA, and mass spectroscopy, they are not as definitive as the methods used for small molecule drugs. Hence, the test for similarity is not as well defined in the case of proteins. However, as... 

Binding of pyridoxal phosphate to peptide PP-42 also appears to be selective for lysine 30. As was indicated by NMR spectroscopy and UV/vis experiments, only one of three potential lysine Schiff bases appeared to form. To determine the site or sites of attachment, the aldimine peptide intermediates were reduced, proteolytically cleaved, and the fragments analyzed by mass spectroscopy. This... 

In another study conducted on marine zooplankton (Byun et ah, 2009b), antioxidant activity was measured for the DPPH radical of hydrolysates produced by Alcalase, a-chymotrypsin, Neutrase, papain, pepsin, and trypsin. To identify antioxidant peptides, peptic hydrolysate was purified using consecutive chromatographic methods, and antioxidant peptides were identified to be Leu-Leu-Gly-Pro-Gly-Leu-Thr-Asn-His-Ala (MW 1076Da) and Asp-Leu-Gly-Leu-Gly-Leu-Pro-Gly-Ala-His (MW 1033Da) by Q-TOF ESI mass spectroscopy. IC50 values of purified peptides were... 

Mass spectroscopy (MS) methods can be used to analyze complex mixtures of proteins and peptides in minutes and with mass accuracy several orders of magnitude better than that obtained from electrophoretic methods. For proteins with a known sequence, the mass measurement accuracies are generally sufficient to identify the products resistant to proteolysis precisely and to define compact domains within proteins effectively. 

The peptide resin prepared above was treated with a 20-fold excess of anhydrous hydrazine in DMF (20 ml) at laboratory temperature for 24 hours, and the mixture was filtered and evaporated to dryness. This procedure also removed the BrZ protecting group from the Tyr moeity. The residue was purified by gel filtration on a column (LH 20 Sephadex) using a 20 1 v/v mixture of water and acetic acid as eluant. There was thus obtained Pyr-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg(H+)-Pro-NH-NH2. The structure of which was confirmed by amino acid analysis and mass spectroscopy. 

Synthetic peptides often lack the conformational stability required for a successful drug therefore determination of peptide stability in serum constitutes a powerful and important screening assay for the elimination of unstable peptides in the pipeline of drug development (see Note 1). Peptide stability in serum can rather easily be determined by reverse phase-high-performance liquid chromatography (RP-HPLC) and mass spectroscopy (MS) from both in vitro and in vivo studies. 

Measurement of the proteolytic serum activity should ideally be done at different time intervals. However, studies have demonstrated that an adequate time point for detection of peptide degradation in sterile serum is 1 h. This time point gave almost full degradation of the unstable control peptide, with comparable levels of the degradation products with both RP-HPLC and mass spectroscopy (15). 

After purification, lyophilize the peptide constructs and check their purity on analytical HPLC and MALDI-TOF mass spectroscopy. The conventional matrix for peptide mass spectroscopy, a-cyano-4-hydroxycinnamic acid, appears to work properly for the vaccine constructs. While it is less frequently used in standard peptide chemistry (and thus not detailed here), the size of these multiple peptide antigens allows quality control by SDS-PAGE as shown in Fig. 2. For this purpose we recommend the use of 16.5% precast peptide gels. These gels tend to break easily, so be careful during gel handling. 

OBOC combinatorial bead-libraries can be considered as chemical microarrays that are spatially separable but non-addressable. The identity of the chemical compound on the positive beads can be determined directly with an automatic sequencer if it is an N-terminally unprotected peptide, by mass spectroscopy, or through chemical encoding. i A synthetic scheme for the OBOC library is shown in Figure 2. Using the highly efficient split-mix synthesis method,literally hundreds of thousands to millions of compounds can be prepared within a week. The recent... 

Analysis of Purified Peptide by MALDI-TOF Mass Spectroscopy The purified lyophilized fractions of peptides (and significant side products)... 

In the first studies, antibodies were used to elucidate the dynamic nature of poliovirus [51]. Antibodies were raised against peptides representing VP4 and the N termini of VPl. In the crystal structures of all of the picornaviruses, these termini lie at the capsid-RNA interface and are therefore not exposed to external solvent [27, 46, 52-55]. These antibodies bound to the virus when the particles were heated to 37°C but did not bind when added to virus at room temperature or when the virus was heated to 37°G and then cooled to room temperature. Therefore, although difficult to visualize with the static structure of the capsid, the only explanation for these results is that these buried termini are transiently exposed. This exposure is facilitated by higher temperatures and was proposed to be part of the normal infection process. This idea of dynamic capsid structures was subsequently supported by mass spectroscopy analysis of flock house virus [56] and rhinovirus [57] and by a series of drug—poliovirus structures [58]. 

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