Reversed-phase Chromatography Fractionation Research Articles

Identification of Antioxidant Peptides in Enzymatic Hydrolysates of Carp (Cyprinus Carpio) Skin Gelatin

The protein by-products from carp (Cyprinus carpio) are normally discarded as industrial waste during fish processing. The objective of this study was to identify and characterise the peptides with a potential antioxidant activity that are released from carp skin proteins during hydrolysis by the Protamex enzyme mixture. This study shows that a hydrolysate of carp skin gelatin and its reversed-phase chromatography fractions have strong in vitro antioxidant properties. Among these fractions, the alanine-tyrosine (Ala-Tyr) dipeptide was identified as the major compound with high antioxidant potential. The peptide has good stability during in vitro enzymatic digestion assay and can inhibit the angiotensin-converting enzyme (ACE). In conclusion, our study proves that both the unfractionated hydrolysate of carp skin gelatin and the above-mentioned Ala-Tyr dipeptide represents attractive novel compounds for the formulation of antioxidant foods.

Proteomic analysis of protein deamidation.

Deamidation of asparagines and glutamines occurs spontaneously in proteins and results in protein degradation. Deamidation of asparaginyl residues in proteins produces a mixture of asparaginyl, n-aspartyl, and isoaspartyl residues, which has been linked to the pathology of some neurodegenerative diseases. However, accurate proteomic analysis of deamidation is challenging since it occurs quickly during conventional proteomic sample preparation, and the co-elution of the two resulting isomeric deamidated peptides in reversed-phase liquid chromatography (RPLC) compromises their identification and quantification using RPLC-MS/MS. To overcome these difficulties, a novel sample preparation protocol to minimize artificial deamidation has been developed alongside an offline RP-ERLIC-MS/MS (reversed-phase chromatography fractionation followed by electrostatic repulsion-hydrophilic interaction chromatography coupled with MS/MS) strategy to separate and quantify the three deamidation products from the same peptide on a proteome-wide scale. These protocols are detailed in this unit.

Sialylated Lewis x Antigen Bearing Glycoproteins in Human Plasma

Recent studies have shown that antibodies targeting Lewis x (Le(x)) antigen are a valuable tool in the isolation and identification of glycoproteins in plasma. A focus of this study was to determine whether sialylated Lewis x (sLe(x)) antigen carrying glycoproteins occur in human plasma and whether an antibody targeting this antigen could be used to isolate and identify glycoproteins bearing this antigen. An additional objective was to determine the degree to which proteins conjugated to Le(x) and sLe(x) antigens are similar in structure. A specific anti-sLe(x) antibody (anti-sLe(x)Ab), CHO-131, immobilized in an immunoaffinity column was used to select a set of specific sLe(x) bearing proteins from human plasma, after which they were identified by either of two analytical strategies. One approach was to further resolve the affinity selected proteins by reversed phase chromatography (RPC), tryptic digest the RPC fractions, and identify peptide fragments by MALDI-MS/MS. The second was to tryptic digest the affinity selected protein fraction, further resolve the tryptic fragments by RPC, and identify peptides from RPC fractions by MALDI-MS/MS. Histidine-rich glycoprotein, plasminogen, apolipoprotein A-I, vitronectin, proteoglycan-4, clusterin, Ig gamma-2 chain C region, Ig mu chain C region, and interalpha-trypsin inhibitor heavy chain H4 were found to change three folds or more in association with breast cancer. Fifty percent of the glycoproteins carrying either sLe(x) antigen from CHO-131 selection, Le(x) antigen from selection with TG-1 antibody, or both were found to be changed three folds or more in concentration in breast cancer plasma relative to controls.

Use of Multidimensional Lectin Affinity Chromatography in Differential Glycoproteomics

This paper reports studies comparing the relative degree of sialylation among human serum glycoproteins carrying complex biantennary N-linked, hybrid, and high-mannose oligosaccharides. Comparisons were made by coupling lectin affinity selection with stable isotope coding of peptides from tryptic digests of serum. After proteolysis, samples were split and differentially acetylated with stable isotope coding agents according to either origin or the separation method by which they would be fractionated. A lectin column prepared from Sambucus nigra agglutinin (SNA) was used to select and compare the concentration of sialic acid containing glycopeptides. The relative standard deviation in quantification using this method was 4%. Using this method the concentration of sialic acid containing glycoproteins from a normal individual were compared to those in a pooled serum sample from a large number of normal individuals. It was found that sialylation varied less than 2-fold in all but four or five glycoproteins. Further studies were done on the degree of sialylation within glycoproteins. Samples labeled with the light isoform of the coding agent were applied to a set of serial lectin columns consisting of a concanavalin A (Con A) column coupled to an SNA column for selecting sialic acid appended to glycopeptides with complex biantennary N-linked, hybrid, and high-mannose glycans. In contrast, samples labeled with the heavy isoform of the coding agent were applied to a Con A lectin column alone to select glycopeptides containing complex biantennary N-linked, hybrid, and high-mannose glycans, without regard to sialylation. Glycopeptides thus selected were mixed, deglycosylated by PNGase F, and fractionated by reversed-phase chromatography (RPC). The RPC fractions were then analyzed by ESI-MS. The relative standard deviation of the method was 4%. All glycopeptides identified contained sialic acid except one. Peptides in which the relative abundance of isotopic isoforms was equal were considered to indicate that the protein parent was fully sialylated at that specific glycosylation site.

Studies of Molecular Association of Glutenins and Maltodextrin in Solution by Capillary Electrophoresis

Molecular associations of wheat glutenins and carbohydrates are important in bread systems and believed to contribute to differences of a number of dough and baking quality characteristics. In this investigation, the study of molecular association of glutenins with carbohydrates in solution was performed by capillary zone electrophoresis (CZE). The carbohydrate used was a maltodextrin of average molecular weight 2000. To follow the molecular association of major glutenin subunits with the maltodextrin of interest, the extracted proteins from hard red winter wheat (cv. Scout 66) were fractionated first by reversed‐phase chromatography (RPC). Four major RPC fractions were collected, each of which still exhibited more than one glutenin component when reanalyzed by CZE. These RPC fractions were then incubated with the maltodextrin of interest, and the pre‐equilibrated glutenin‐maltodextrin complexes thus obtained were analyzed by CZE. Since maltodextrin is a neutral polymer, its binding to the glutenins results in decreasing the charge‐to‐mass ratio of the glutenins, and consequently, the effective electrophoretic mobility of protein–maltodextrin complex decreases with increasing maltodextrin concentration in the incubation solution. The evaluation of glutenin–maltodextrin interactions was only qualitative and the calculation of binding constants was hampered by various factors including the heterogeneity of the glutenin RPC fractions. The components of each RPC fraction yielded mobility lines (i.e., plots of electrophoretic mobility vs. percentage of maltodextrin in the glutenin sample) that paralleled each other indicating that these components have the same binding energetics magnitude with maltodextrin. This observation was substantiated by the sodium dodecyl sulfate capillary gel electrophoresis (SDS‐CGE) analysis, which showed that the various components of each RPC fraction are probably formed from one kind or one group of glutenin subunit with very similar properties. In addition, the mobility curves did not exhibit the usual rectangular hyperbolic forms of 1:1 binding isotherms.