Contaminating peptides

In addition to the precautions already mentioned, the time of column equilibration between each run should be about the same. Ideally the profile of one of the two peptides (native or synthetic) should give a symmetrical absorbance as well as show the presence of a small amount of an impurity. Under such circumstances, the coelution run should show a symmetrical absorbance of the size of that of the synthetic and native molecules. In addition, the absorbance of the impurity should now be half the size of that seen in the chromatogram of the contaminated peptide. All other absorbances such as injection artifacts should be identical in all runs 2-6. CAUTION Two peptides that show the same retention times after being run separately under the same conditions cannot be said to have coeluted ... 

Boil a few mg each of the product and of ninhydrin (Expt 5.99) in 0.5 ml of water the absence of a blue-purple coloration shows that the product is free from contaminating peptide material. 

Fig. 2. SDS-PAGE of the Fmoc-protected original M2e-Sl-S2 dimeric construct used for immunization after deprotection (14). The left lane shows the molecular weight standards (the bands correspond to 42, 30, and 22 kDa, from the top to the bottom, respectively) the right lane documents the purity of the final product (calculated molecular weight 18,434 Da). While the general quality of the peptide for its size is excellent, insufficient dimer formation and the presence of contaminating peptides can be observed.

If the identity of the OP-labeled protein is unknown, a tagged OP, for example a biotinylated OP, can be used to identify the protein (Schopfer et ah, 2005). After the identity of the OP-labeled protein is known, identification of the OP-labeled peptide depends on separating it from contaminating peptides. We have found that the OP-labeled peptide is frequently not found by mass spectrometry unless it has been extensively purified. In some cases it is possible to identify the labeled peptide simply by LC-MS-MS, where an enzymatic digest of the isolated protein is subjected to liquid chromatography on a Cl8 nanocolumn and the effluent from the column is electrosprayed directly into the mass spectrometer. For other cases, extensive HPLC puri-flcation of the enzymatic digest is necessary to obtain a purifled fraction of peptides that can be introduced into the mass spectrometer. 

In this paper, we report the use of several micro-porous synthetic membranes to prepare contaminated peptides and proteins for MALDl-TOF analysis. By spotting contaminated samples directly onto activated synthetic membranes, impurities such as salts, glycerol, and detergents can be washed from the sample, while biopolymers remain intact. Following addition of matrix, samples can be desorbed and ionized... 

Worrall, T. A., Cotter, R. J., and Woods, A. S., Purification of contaminated peptides and proteins on synthetic membrane surfaces for matrix-assisted laser/desorption ionization mass spectrometry. Analytical Chemistry, 70, 750-756, 1998. 

Under optimized measurement conditions, with low laser power in linear negative ion mode, complete sulfate retention was routinely observed in the most abundant ion. However, some loss of sulfate was generally observed, giving rise to additional signals that could be mistaken for contaminating peptide species. Hence, it is crucial to use RP-HPLC-purified sulfotyrosine peptides for MALDI-TOF MS analysis. For a more detailed discussion of the characteristic loss-of-sulfate peak patterns observed in MALDI-TOF mass spectra of sulfotyrosine peptides, we refer to the following references Seibert et al. (2002, 2008) and Seibert and Sakmar (2008). 

Solid phase peptide synthesis does not solve all purification problems however Even if every coupling step m the ribonuclease synthesis proceeded in 99% yield the product would be contaminated with many different peptides containing 123 ammo acids 122 ammo acids and so on Thus Memfield and Gutte s six weeks of synthesis was fol lowed by four months spent m purifying the final product The technique has since been refined to the point that yields at the 99% level and greater are achieved with current instrumentation and thousands of peptides and peptide analogs have been prepared by the solid phase method... 

These authors also mention some shortcomings that should be borne in mind, in particular, that some peptides observed were from the autolysis of trypsin, the digestion agent, and from contaminants such as human keratin, while some peptide ions did not produce interpretable MS-MS spectra. 

Recently, unique vesicle-forming (spherical bUayers that offer a hydrophilic reservoir, suitable for incorporation of water-soluble molecules, as well as hydrophobic wall that protects the loaded molecules from the external solution) setf-assembUng peptide-based amphiphilic block copolymers that mimic biological membranes have attracted great interest as polymersomes or functional polymersomes due to their new and promising applications in dmg delivery and artificial cells [ 122]. However, in all the cases the block copolymers formed are chemically dispersed and are often contaminated with homopolymer. 

In contrast to these results, Ross et al. [33] found that antisera against a 20-amino acid peptide (Ser-613-Arg-632) of the cytoplasmic domain of the human Na /H exchanger recognized a 66-kDa protein in immunoblots of bovine renal brush border membranes. Since the purity of these membranes was not reported it is possible that this result was due to contamination with basolateral membranes (although the molecular mass would still differ from the basolateral Na /H exchanger in LLC-... 

For some downstream applications of the purified protein, the contaminating 3XFLAG peptide (mol. wt. 2862) must be removed. This is achieved by dialyzing with an appropriate buffer. 

The type of proteinaceous binder was correctly identified in all model samples. In only one case (S10), the animal glue was additionally identified, although the restorer who prepared these model samples declared that the sample contained only egg binder. It is possible that this sample was contaminated during its preparation or during laboratory treatment. The results indicate that this method does not allow reliable identification of the presence of individual egg yolk and egg white most probably it is caused by the presence of a trace of egg white that is always present in the egg yolk preparations (and vice versa) and can be detected by the highly sensitive PMM method. The identification of individual types of animal glues will never be reliable by MALDI-TOF mass spectrometry because of their similar composition the application of ESI (electrospray ionisation)-MS/MS (Section 6.5) could possibly overcome this problem. Only the fish glue, whose peptide... 

Figure 17.27 The EPL process involves a fusion protein containing an intein tag plus a CBD. The fusion protein is captured on an immobilized chitin resin and after removal of contaminating proteins, it is eluted using thiophenol, which cleaves at the thioester bond between the intein and the desired expressed protein. This releases a phenylth-ioester-activated protein that can be used in the native chemical ligation reaction with another peptide containing an N-terminal cysteine residue. Conjugation results in a native amide (peptide) bond formed between them.

Figure 19.19 shows a plot of the results of such an assay done to determine the maleimide content of activated BSA. This particular assay used 2-mercaptoethanol which is relatively unaffected by metal-catalyzed oxidation. For the use of cysteine or cysteine-containing peptides in the assay, however, the addition of EDTA is required to prevent disulfide formation. Without the presence of EDTA at 0.1 M, the metal contamination of some proteins (especially serum proteins such as BSA) is so great that disulfide formation proceeds preferential to maleimide coupling. Figure 19.20 shows a similar assay for maleimide-activated BSA using the more innocuous cysteine as the sulfhydryl-containing compound. 

Fig. 10 Representative solid-state 19F-NMR spectra of 19F-labelled gramicidin S (substituted at both Leu positions with 4F-Phg), embedded in macroscopically oriented bacterial protoplasts at a peptide-to-lipid ratio of about 1 40. These membrane samples are intrinsically less well oriented than the ghosts in Fig. 8, and a TFA contamination is marked with an asterisk...

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