We present an improved protocol for coupling synthetic peptides to carrier proteins. In this prot... more We present an improved protocol for coupling synthetic peptides to carrier proteins. In this protocol, dimethyl-formamide is used as the solvent to solubilize peptides instead of phosphate-buffered saline (PBS) or 6 M guanidine-HCl/0.01 M phosphate buffer (pH 7). Additionally, the last desalting or dialyzing step to remove uncoupled peptides as in the traditional method is eliminated. Finally, 3 ml of 0.1 M ammonium bicarbonate is added to the carrier protein conjugated peptide solution to help the lyophilization process. Coupling of Cys-containing synthetic peptides to keyhole limpet hemocyanin or bovine serum albumin using m-maleimidobenzoyl-N-hydroxysuccinimide ester are used as the test cases. This method produces high-quality antipeptide antibodies. Also, compared to the traditional method, this procedure is simpler and useful for peptides with solubility problems in PBS or 6 M guanidine-HCl.
MALDI-TOF MS is used successfully in investigating in vitro glycation of normal and variant hemog... more MALDI-TOF MS is used successfully in investigating in vitro glycation of normal and variant hemoglobins (Hbs). Singly glycated, doubly glycated, and/or multiply glycated glycoisoforms of the alpha-globin, beta-globin, and gamma-globin of Hbs are observed. Different glycation rates are observed for normal and variant Hbs, with the normal Hb A having the slowest rate. The normal Hb A is more stable than the Hb C, Hb E, Hb F, Hb Leiden, and Hb San Diego upon condensation with glucose at 37°C. Data reveal that with longer incubation time (up to 5 d), higher glucose concentration (up to 1 M), and higher temperature (up to 37°C), the number of glycated amino acid residues of Hbs increase. The extent of the glycation of both Hb A and Hb F increases upon changing the solvent from PBS (pH 7.4) to carbonate buffer (pH 10). However, this pH change has a lesser effect on the glycation of the Hb C, Hb E, or Hb Leiden. In this study, higher concentration of the glucose is used to increase the rea...
Drug metabolism and pharmacokinetics (DMPK) studies require the identifi cation of drug metabolit... more Drug metabolism and pharmacokinetics (DMPK) studies require the identifi cation of drug metabolites usually by mass spectrometry (MS), and in later stages of drug development, confi rmation of metabolite structures by NMR. To achieve this, purification of metabolites may be required. In this Application Note, purification of bupropion drug metabolites was achieved using Agilent Automated Purification Software in conjunction with the Agilent 1260 Analytical-scale Purification System. The metabolites were identifi ed using high-resolution accurate mass MS and MS/MS data acquired using an Agilent 6540 Accurate Mass Quadrupole Time-of-Flight (Q-TOF) LC/MS system and Mass-MetaSite Software.
This Technical Overview highlights some of the added features of GeneSpring 13 (GS 13) as applied... more This Technical Overview highlights some of the added features of GeneSpring 13 (GS 13) as applied to retinoblastoma, a childhood eye cancer affecting children usually less than 5 years of age. Cancer development is driven by inactivation of both copies of the RB1 gene in a child’s retina. In this study we looked into the transcriptomic profiling of mRNA and miRNA of human retinoblastomas.
Biocytin hydrazide is widely used to biotinylate the carbohydrate moieties of glycoproteins. In t... more Biocytin hydrazide is widely used to biotinylate the carbohydrate moieties of glycoproteins. In this study, however, biocytin hydrazide was found to be able to directly biotinylate peptides and proteins. This phenomenon may cause false identification of non-glycopeptides/non-...
We present an improved protocol for coupling synthetic peptides to carrier proteins. In this prot... more We present an improved protocol for coupling synthetic peptides to carrier proteins. In this protocol, dimethyl-formamide is used as the solvent to solubilize peptides instead of phosphate-buffered saline (PBS) or 6 M guanidine-HCl/0.01 M phosphate buffer (pH 7). Additionally, the last desalting or dialyzing step to remove uncoupled peptides as in the traditional method is eliminated. Finally, 3 ml of 0.1 M ammonium bicarbonate is added to the carrier protein conjugated peptide solution to help the lyophilization process. Coupling of Cys-containing synthetic peptides to keyhole limpet hemocyanin or bovine serum albumin using m-maleimidobenzoyl-N-hydroxysuccinimide ester are used as the test cases. This method produces high-quality antipeptide antibodies. Also, compared to the traditional method, this procedure is simpler and useful for peptides with solubility problems in PBS or 6 M guanidine-HCl.
A method, which utilizes microwave-assisted partial acid hydrolysis and matrix-assisted laser des... more A method, which utilizes microwave-assisted partial acid hydrolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), to elucidate oligosaccharide composition of intact glycoproteins is presented here. Glycoproteins, such as ribonuclease B, avidin, alpha1-acid glycoprotein, and fetuin, are used as model systems to demonstrate this technique. Partial cleavage of oligosaccharides from whole intact glycoproteins with trifluoroacetic acid was observed after a short exposure to microwaves. Due to the high-resolution mass spectra obtained by MALDI-TOFMS from glycoproteins with molecular weights less than 20 kDa, the compositions of oligosaccharides are readily derived for ribonuclease B and avidin. The data agree with the proposed oligosaccharide structures of ribonuclease B (five glycoforms) and avidin (eight glycoforms). Larger glycoproteins such as alpha1-acid glycoprotein (many glycoforms) and fetuin (many glycoforms) exhibited only broad peaks with no glycoform resolution. Nevertheless, this method can be used successfully for analysis of glycoproteins with molecular weights greater than 20 kDa to determine the presence or absence of glycosylation.
The phenomenon of protein randomly associating among themselves during MALDI-TOF mass spectrometr... more The phenomenon of protein randomly associating among themselves during MALDI-TOF mass spectrometric measurements is manifest on all the proteins tested here. The magnitude of this random association seems to be protein-dependent. However, a detailed mathematical analysis of this process has not been reported so far. Here, binomial and multinomial equations are used to analyze the relative populations of multimer ions
We present an improved protocol for coupling synthetic peptides to carrier proteins. In this prot... more We present an improved protocol for coupling synthetic peptides to carrier proteins. In this protocol, dimethyl-formamide is used as the solvent to solubilize peptides instead of phosphate-buffered saline (PBS) or 6 M guanidine-HCl/0.01 M phosphate buffer (pH 7). Additionally, the last desalting or dialyzing step to remove uncoupled peptides as in the traditional method is eliminated. Finally, 3 ml of 0.1 M ammonium bicarbonate is added to the carrier protein conjugated peptide solution to help the lyophilization process. Coupling of Cys-containing synthetic peptides to keyhole limpet hemocyanin or bovine serum albumin using m-maleimidobenzoyl-N-hydroxysuccinimide ester are used as the test cases. This method produces high-quality antipeptide antibodies. Also, compared to the traditional method, this procedure is simpler and useful for peptides with solubility problems in PBS or 6 M guanidine-HCl.
MALDI-TOF MS is used successfully in investigating in vitro glycation of normal and variant hemog... more MALDI-TOF MS is used successfully in investigating in vitro glycation of normal and variant hemoglobins (Hbs). Singly glycated, doubly glycated, and/or multiply glycated glycoisoforms of the alpha-globin, beta-globin, and gamma-globin of Hbs are observed. Different glycation rates are observed for normal and variant Hbs, with the normal Hb A having the slowest rate. The normal Hb A is more stable than the Hb C, Hb E, Hb F, Hb Leiden, and Hb San Diego upon condensation with glucose at 37°C. Data reveal that with longer incubation time (up to 5 d), higher glucose concentration (up to 1 M), and higher temperature (up to 37°C), the number of glycated amino acid residues of Hbs increase. The extent of the glycation of both Hb A and Hb F increases upon changing the solvent from PBS (pH 7.4) to carbonate buffer (pH 10). However, this pH change has a lesser effect on the glycation of the Hb C, Hb E, or Hb Leiden. In this study, higher concentration of the glucose is used to increase the rea...
Drug metabolism and pharmacokinetics (DMPK) studies require the identifi cation of drug metabolit... more Drug metabolism and pharmacokinetics (DMPK) studies require the identifi cation of drug metabolites usually by mass spectrometry (MS), and in later stages of drug development, confi rmation of metabolite structures by NMR. To achieve this, purification of metabolites may be required. In this Application Note, purification of bupropion drug metabolites was achieved using Agilent Automated Purification Software in conjunction with the Agilent 1260 Analytical-scale Purification System. The metabolites were identifi ed using high-resolution accurate mass MS and MS/MS data acquired using an Agilent 6540 Accurate Mass Quadrupole Time-of-Flight (Q-TOF) LC/MS system and Mass-MetaSite Software.
This Technical Overview highlights some of the added features of GeneSpring 13 (GS 13) as applied... more This Technical Overview highlights some of the added features of GeneSpring 13 (GS 13) as applied to retinoblastoma, a childhood eye cancer affecting children usually less than 5 years of age. Cancer development is driven by inactivation of both copies of the RB1 gene in a child’s retina. In this study we looked into the transcriptomic profiling of mRNA and miRNA of human retinoblastomas.
Biocytin hydrazide is widely used to biotinylate the carbohydrate moieties of glycoproteins. In t... more Biocytin hydrazide is widely used to biotinylate the carbohydrate moieties of glycoproteins. In this study, however, biocytin hydrazide was found to be able to directly biotinylate peptides and proteins. This phenomenon may cause false identification of non-glycopeptides/non-...
We present an improved protocol for coupling synthetic peptides to carrier proteins. In this prot... more We present an improved protocol for coupling synthetic peptides to carrier proteins. In this protocol, dimethyl-formamide is used as the solvent to solubilize peptides instead of phosphate-buffered saline (PBS) or 6 M guanidine-HCl/0.01 M phosphate buffer (pH 7). Additionally, the last desalting or dialyzing step to remove uncoupled peptides as in the traditional method is eliminated. Finally, 3 ml of 0.1 M ammonium bicarbonate is added to the carrier protein conjugated peptide solution to help the lyophilization process. Coupling of Cys-containing synthetic peptides to keyhole limpet hemocyanin or bovine serum albumin using m-maleimidobenzoyl-N-hydroxysuccinimide ester are used as the test cases. This method produces high-quality antipeptide antibodies. Also, compared to the traditional method, this procedure is simpler and useful for peptides with solubility problems in PBS or 6 M guanidine-HCl.
A method, which utilizes microwave-assisted partial acid hydrolysis and matrix-assisted laser des... more A method, which utilizes microwave-assisted partial acid hydrolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), to elucidate oligosaccharide composition of intact glycoproteins is presented here. Glycoproteins, such as ribonuclease B, avidin, alpha1-acid glycoprotein, and fetuin, are used as model systems to demonstrate this technique. Partial cleavage of oligosaccharides from whole intact glycoproteins with trifluoroacetic acid was observed after a short exposure to microwaves. Due to the high-resolution mass spectra obtained by MALDI-TOFMS from glycoproteins with molecular weights less than 20 kDa, the compositions of oligosaccharides are readily derived for ribonuclease B and avidin. The data agree with the proposed oligosaccharide structures of ribonuclease B (five glycoforms) and avidin (eight glycoforms). Larger glycoproteins such as alpha1-acid glycoprotein (many glycoforms) and fetuin (many glycoforms) exhibited only broad peaks with no glycoform resolution. Nevertheless, this method can be used successfully for analysis of glycoproteins with molecular weights greater than 20 kDa to determine the presence or absence of glycosylation.
The phenomenon of protein randomly associating among themselves during MALDI-TOF mass spectrometr... more The phenomenon of protein randomly associating among themselves during MALDI-TOF mass spectrometric measurements is manifest on all the proteins tested here. The magnitude of this random association seems to be protein-dependent. However, a detailed mathematical analysis of this process has not been reported so far. Here, binomial and multinomial equations are used to analyze the relative populations of multimer ions
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Papers by Syed Salman Lateef