Identification of Glycosylation

To detect the presence of glycosylation, two approaches are in common use comparative peptide mapping and selective detection of glycopeptides. 

Seiective Detection by Precursor-Ion Scan The precursor-ion scan of specific marker ions (e.g., m/z 204) is another option for selective detection of glycopeptides [77], For example, the MS-2 of a tandem instrument can be set to monitor m/z 204, and the MS-1 is scanned to detect those glycopeptides that 

Once glycopeptides have been detected, the corresponding RP-HPLC fractions are collected and treated further with exoglycosidases to release specific sugar residues. The molecular mass is determined again, and the difference in the two masses provides the identity of the carbohydrate residue that was attached 

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Caro, L., Tettelin, H., Vossen, J., Ram, A., van den Ende, H., and Klis, F. (1997). In silico identification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisiae. Yeast 13, 1477—1489. 

Identification of glycosyl-enzyme intermediates. Studies with pure enzymes often make it possible to confirm directly the existence of enzyme-bound intermediates. The intermediates detected are frequently glycosyl esters of glutamate or aspartate side chain... 

Positive Identification of Glycosylation Sites In Proteins And Peptides Using A Modified Beckman LF 3600 N-Terminal Protein Sequencer... 

Several examples using sequencing techniques developed for the positive identification of glycosylation sites of proteins and peptides are... 

An example for the separation for flavonoids with HP-RPC is the screening method employed for the systematic identification of glycosylated flavonoids and other phenolic compounds in plant food materials by Lin et al20 These authors used an analytical 4.6 mm x 250 mm 5 pm C18 silica column at 25 °C with linear gradient elution (eluent A (0.1% FA in water and eluent B 0.1% FA in ACN) at 1.0 ml min-1. DAD was performed at 270, 310, 350, and 520 nm to monitor the UV/VIS absorption. The LC system was directly coupled to an ESI mass spectrometer without flow splitting and the mass spectra acquired in the positive and negative ionization mode. The same analytical scheme (aqueous MeOH extraction, reversed-phase liquid chromatographic separation, and diode array and mass spectrometric detection) can be applied to a wide variety of samples and standards and therefore allows the cross-comparison of newly detected compounds in samples with standards and plant materials previously identified in the published literature. 

Ito, A., T. A. Okamura, K. Uegaki et al. 2009. Mass spectrometric analysis using ruthenium (Il)-labeUing for identification of glycosyl hydrolase product. Biosci. Biotechnol. Biochem. 73 428-430. 

A fast micromcthod (10-15 nmol scale) for the identification of glycosyl phosphate anomers based on spectrophotometric measurement of the rate of dieir acid-catalysed hydrolysis has been described. The standard Gibbs free-energy change for hydrolysis of a-D-ribose 1-phosphate has been determined. 

Lin L-Z, Hamly JM (2007) A screening method for the identification of glycosylated flavonoids and other phenolic compounds using a standard analytical approach for all plant materials. J Agrlc Food Chem 55 1084—1096. doi 10.1021/jf062431s... 

DNA sequencing reveals the order in which amino acids are added to the nascent polypeptide chain as it is synthesized on the ribosomes. However, it provides no information about posttranslational modifications such as proteolytic processing, methylation, glycosylation, phosphorylation, hydroxylation of prohne and lysine, and disulfide bond formation that accompany mamra-tion. While Edman sequencing can detect the presence of most posttranslational events, technical hmitations often prevent identification of a specific modification. 

Fig. 3. (A) Disposition of afi unit in the membrane, based on sequence information [14,15], selective proteolytic digestion of the a subunit [5,6] and hydrophobic labelling (Table 1). The model for the (S subunit is based on sequencing of surface peptides and identification of S-S bridges [64,65]. T, T2 and C3 show location of proteolytic splits. CHO are glycosylated asparagines in the P subunit. (B) Peptide fragments remaining in the membrane after extensive tryptic digestion of membrane-bound Na,K-ATPase from outer medulla of pig kidney as described by Karlish et al. [7,58].

Post-translational modification of proteins plays a critical role in cellular function. For, example protein phosphorylation events control the majority of the signal transduction pathways in eukaryotic cells. Therefore, an important goal of proteomics is the identification of post-translational modifications. Proteins can undergo a wide range of post-translational modifications such as phosphorylation, glycosylation, sulphonation, palmitoylation and ADP-ribosylation. These modifications can play an essential role in the function of the protein and mass spectrometry has been used to characterize such modifications. 

S. Kazuno, M. Yanagida, N. Shindo and K. Murayama, Mass spectrometric identification and quantification of glycosyl flavonoids, including dihydrochalcones with neutral loss scan mode, Anal. Biochem., 347, 182 192 (2005). 

Wells-Knecht KJ, Zyzak DV, Litchield JE, Thorpe SR and Baynes JW (1995a) Mechanism of autoxidative glycosylation identification of glyoxal and arabi-nose as intermediates in the autoxidative modification of proteins by glucose. Biochemistry 34, 3702-3709. 

There have been many survey studies of citrus flavonoids, yet by no means has an exhaustive analysis been performed for all species and cultivars. Surveys often are initiated by hydrolysis of tissue extracts and identification of the flavonoid aglycones present in the tissues. This provides a basis for subsequent identification of the glycosylated compounds present... 

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