Peptides glycopeptide separation

For most free amino acids and small peptides, a mixture of alcohol with water is a typical mobile phase composition in the reversed-phase mode for glycopeptide CSPs. For some bifunctional amino acids and most other compounds, however, aqueous buffer is usually necessary to enhance resolution. The types of buffers dictate the retention, efficiency and - to a lesser effect - selectivity of analytes. Tri-ethylammonium acetate and ammonium nitrate are the most effective buffer systems, while sodium citrate is also effective for the separation of profens on vancomycin CSP, and ammonium acetate is the most appropriate for LC/MS applications. 

Carbohydrates and polysaccharides, on the one hand, and peptides and proteins, on the other, have been considered as separate classes of natural products for a long time. Fundamental chemical methodology for the synthesis of both saccharides and peptides was developed by Emil Fischer et al. at the beginning of the 20th century. 1,2 However, the harsh conditions employed in early solution and solid-phase peptide synthesis hindered the combination of peptide and carbohydrate chemistry, i.e. glycopeptide synthesis. Considerable efforts were made to combine the two branches of natural product chemistry, and the state of the art within glycopeptide synthesis has improved dramatically during the last decades, as described in a number of reviews. 3 23,512"514 ... 

Analytical Properties Substrate has 38 chiral centers and 7 aromatic rings surrounding 4 cavities (A, B, C, D), making this the most structurally complex of the macrocyclic glycopeptides substrate has a relative molecular mass of 2066 this phase can be used in normal, reverse, and polar organic phase separations selective for anionic chiral species with polar organic mobile phases, it can be used for a-hydroxy acids, profens, and N-blocked amino acids in normal phase mode, it can be used for imides, hydantoins, and N-blocked amino acids in reverse phase, it can be used for a-hydroxy and halogenated acids, substituted aliphatic acids, profens, N-blocked amino acids, hydantoins, and peptides Reference 47, 48... 

Glycopeptides from a peptide mixture were analyzed via normal phase IPC, using inorganic monovalent ions as IPRs (added only to the sample and not to the mobile phase) to increase the hydrophobicity differences among peptides and glycopeptides [63]. IPC separation of peptides usually relies on the use of perlluorinated carboxylic acids [64] and small chaotropes [65,66], It can be performed also on a preparative scale for the purification of peptides from natural sources [67], Amino acids and related compounds [68-72] were similarly analyzed via IPC-MS. 

Table 1 summarizes the carbohydrate structural data deduced from the three detection methodologies, MALDI-TOF, ES-MS, and high pH anion exchange chromatography. The N-glycosylation site proved to be occupied with several structures. The glycopeptides were partially separated as five peptides, at retention times of 19.4, 20.3, 20.7, 20.9, and 21.3 minutes, and all of these glycopeptides contained the same amino acid sequence, N-terminal amino acids 1-17. 

Due to an increased interest in analysis of physiological samples, we wanted to establish analyzer methods which would allow us to choose between our standard protocol for protein and peptide hydrolysates and a separate protocol for an expanded number of amino acids, to include the most important free amino acids found in physiological samples. A study of common analysis requirements in our facility indicated that only a limited number of the possible free physiological amino acids is needed for most unknown samples. These additional amino acids of interest are a-amino butyric acid, citrulline, y-amino butyric acid (GABA), hydroxyproline, hydroxylysine, ornithine, taurine, and tryptophan. Other amino acids of interest to us are phosphoserine, phosphothreonine, phosphotyrosine and carboxy-amino acids since they are released from glycoprotein or glycopeptide hydrolysates. 

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