Peptides ultraviolet detection

Detection in 2DLC is the same as encountered in one-dimensional HPLC. A variety of detectors are presented in Table 5.2. The choice of detector is dependent on the molecule being detected, the problem being solved, and the separation mode used for the second dimension. If MS detection is utilized, then volatile buffers are typically used in the second-dimension separation. Ultraviolet detection is used for peptides, proteins, and any molecules that contain an appropriate chromophore. Evaporative light scattering detection has become popular for the analysis of polymers and surfactants that do not contain UV chromophores. Refractive index (RI) detection is generally used with size exclusion chromatography for the analysis of polymers. 

Stehle, P., and Rtirst, P. (1985). Isotachophoretic control of peptide-synthesis and purification - a novel-approach using ultraviolet detection at 206 nm.. Chromatogr. 346, 271—279. 

Peptides are commonly detected by absorbance at 200-220 nm. However, most of the compounds present in wine may interfere in the ultraviolet detection of peptides when low wavelengths are used. Thus, for the analysis of these compounds it is useful to apply sensitive and selective detection methods. To this end, it is possible to form derivates of the peptides that can be detected at higher and more specific wavelengths. Detection by fluorescence can also be used to detect peptides containing fluorescence amino acids (tyrosine and tryptophan). For peptides without this property, the formation of derivates with derivatizing agents have been proved to be very useful (Moreno-Arribas et al. 1998a). 

Most spider neurotoxins characterized to date have been found to be low molecular weight organic molecules (inorganic ions and salts, free acids and ami-noacids, biogenic amines, neurotransmitters, and acylpolyamines), peptides, or proteins. Polyamines and a variety of polypeptides with a molecular mass ranging from 3000 to 8000 Da appear to represent the main toxic compounds in spiders. More than 60 peptide toxins have been described to date, whose presence can be confirmed by reversed-phase LC. Peptides can be easily distinguished from polyamines because of their different retention times and by ultraviolet detection using a diode-array detector. 

Assays for melanocyte-stimulating hormone were carried out by Mr. S. Kulovich using the in vitro frog skin assay (1). Subcutaneous assay for adrenocorticotropic activity (2), based on ascorbic acid depletion in hypophysectomized rats, was performed by Dr, J.D. Fisher of the Armour Laboratories. Acid hydrolysates (constant boiling HCl, deaerated, 22 hours, 110°) of these fractions were characterized by automatic amino acid analysis with a Spinco model 120B analyzer. The number of tryptophan residues in the intact peptide was estimated from the ultraviolet absorption curves made with a Cary model 15 spectrophotometer. Electrophoresis was carried out on Whatman paper No. 1 with pyridine-acetate buffer, pH 6.5, and 4 molar urea for 3 hours at 26 volts per cm. Peptides were detected with bromphenol blue(3). 

Solinova V, Kasicka V, Koval D et al (2004) Analysis of synthetic derivatives of peptide hormones by capillary zone electrophoresis and micellar electrokinetic chromatography with ultraviolet-absorption and laser-induced fluorescence detection. J Chromatogr B 808 75-82... 

Reaction with phenylisothiocyanate (PITC) in alkaline conditions produces stable phenylthiocarbamyl (PTC) adducts which can be detected either in the ultraviolet below 250 nm or electrochemically. However, this method involves a complex derivatization procedure and offers poorer sensitivity than the alternatives available for individual amino acids. It is useful, however, in conjunction with the automated analysis of peptides when single derivatized residues can be cleaved and analysed after conversion in acidic conditions to phenylthiohydantoins. 

Peptides are usually detected by absorbance at between 200 and 220 rnn at concentrations of between 100 and 1000 ng (80). Since many solvents and even other components in the samples absorb at these wavelengths, samples must be carefully purified, and the solvents used must be transparent to ultraviolet light. Detection of peptides with aromatic amino acids (Phe, Tyr, Trp) can be carried out at 254 rnn if tyrosine or tryptophan is present, detection at 280 nm is feasible. In some cases detection has been carried out at 230 nm (59). 

Although the peptide aminomethylcoumarin (AMC) substrate approach has been widely used for measuring the activities of many proteases, a disadvantage of this approach arises from the ultraviolet excitation and emission wavelengths required for detecting free AMC where there is a possibility for fluorescence interference. RllO-labeled substrates are less encumbered by interference due to a spectral red shift, but many compounds in small molecule chemical libraries exhibit fluorescent properties that may interfere with assays based on fluorescence detection (Simenov et al. 2008). 

Peptide bonds enable proteins and peptides to be directly detected by ultraviolet (UV) radiation, at 200-220 nm, where the absorption is proportional to the number of... 

Second, combined evidence from theoretical computer modeling studies of short peptides (too short to form any detectable a-helix or (3-sheet) in aqueous solution and a variety of spectroscopic studies, including ultraviolet CD (Rucker et al., 2002), nuclear magnetic resonance (NMR) (Poon et al., 2000), two-dimensional vibrational spectroscopy (Woutersen and Hamm, 2001), vibrational circular dichroism (VCD) (Keiderling et al., 1999), and vibrational Raman spectroscopy (Blanch et al., 2000), reveal that the PPII helix is the dominant conformation in a variety of these short peptides. 

Capillary electrophoresis is an excellent microseparation technique that has been used for the separation of a wide diversity of different molecules." Its separation capabilities extend to ions, small molecules (such as amino acids), and large biomolecules (such as peptides, proteins, and nucleic acids). Indeed the human genome project owes its success, in part, to the use of CE for the separation of DNA bases. In the past, CE has been combined with detection devices such as ultraviolet (UV) and laser-induced fluorescence (LIE) spectrophotometers. The detection of the separated analytes is carried out on column by etching the capillary. Unfortunately, UV detection lacks sensitivity and not every compound of interest will absorb in the UV region of the spectmm. Detection using LIE is sensitive, however, the analytes of interest may require derivatization with a fluorescent tag or have an aromatic amino acid in their structure (e.g., proteins and peptides). An advantage of MS detection that neither UV nor LIE detection provides is the information necessary to directly determine the structure of the detected analyte(s). 

Taking this into account, more sensitive methods of sample detection have been developed, for example, special detection cell constructions for ultraviolet (UV) adsorption and laser-induced fluorescence (LIE), and particularly the introduction of mass spectrometry (MS) brought tremendous progress in online and offline characterization not only of modified peptides but also of modified proteins. In this chapter, we try to provide information about capillary electrophoresis (CE) methods developed for the separation of proteins and peptides with various PTMs. 

This sensitivity is superior to that attained by other methods. Postcolumn reaction with o-phthalaldehyde has been used to detect the tryptic peptides generated from several hundred picomoles (10 jig) of human globulin (Benson, 1976). Generally, about 1 /xg of a protein is required for visualizing a band on a polyacrylamide gel by staining with Coomassie blue. Ultraviolet absorption at 210 nm was used by Ling et al. (1976) to monitor the chromatographic purification of about 200 nmol of a-endorphin. 

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