Biomedical subjects

Mehdi Moini

Publications and source records attributed to Mehdi Moini.

9 recordsLinked to original sources

PubMed2006

Analysis of complex protein mixtures with improved sequence coverage using (CE-MS/MS)n.

Identification of proteins, in a complex protein mixture, using one-dimensional high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) analysis of its digest, usually suffers from low sequence coverage. There are several reasons for the low coverage including undersampling, wide concentration dynamic range of the proteins in a complex protein mixture, and wide range of electrospray ionization efficiency of peptides under each mobile-phase composition. To address this low sequence coverage, we introduce a novel technique, (CE-MS/MS)n, which utilizes the most significant advantages of CE-MS/MS, including economy of sample size, fast analysis time, and high separation efficiency, to increase the sequence coverage of a complex protein mixture. Based on these characteristics, (CE-MS/MS)n can be performed in which multiple CE-MS/MS subanalyses (injections followed by analyses) are analyzed and experimental variables are manipulated during each CE-MS/MS subanalysis in order to maximize sequence coverage. (CE-MS/MS)n is a practical technique since each CE-MS/MS subanalysis consumes <10 nL, and each CE-MS/MS subanalysis takes approximately 10 min; therefore, several subanalyses can be performed in approximately 1 h consuming only nanoliters of the sample. Two techniques have been introduced to address the undersampling: (1) (CE-MS/MS)n using dynamic exclusion. In this technique, several CE-MS/MS analyses (injection followed by separation) were performed in one run using the dynamic exclusion capability of the mass spectrometer until all peptide peaks were analyzed by MS/MS. (2) Gas-phase fractionation. In this technique, (CE-MS/MS)n is performed by scanning a narrow mass range (every approximately 100 m/z) during each CE-MS/MS subanalysis without using dynamic exclusion. Under this condition, in each subanalysis, the number of peptides available for MS/MS analysis is significantly reduced, and peptides with the same nominal masses are analyzed, thereby increasing sequence coverage. Additionally, to address the lack of detection of low-level peptides in a mixture containing a wide concentration dynamic range, the concentration of the sample was systematically increased in each subanalysis (while utilizing dynamic exclusion) so that low-intensity peptides would rise above the mass spectrometer threshold and, consequently, undergo MS/MS analysis. Moreover, to alter the ionization efficiency of peptides with low electrospray ionization efficiency, and to change the migration behavior of comigrating peptides under a specific liquid composition, the CE background electrolyte was modified in several subanalyses to further improve sequence coverage. The combination of the above-mentioned techniques was applied to the analysis of the tryptic digests of three well-characterized protein mixtures: a six-protein mixture with average MW of approximately 26,000 (standard I), a six-protein mixture with an average MW approximately 49,000 (standard II), and a more complex protein mixture containing 55 proteins (E. coli ribosomal proteins). In approximately 1 h, when the MS/MS of the peptides were manually checked, all peptides that produced peaks under electrospray ionization in the scanned range of the analysis (500-2000 m/z) and within the practical fragmentation capability of the MS (peptides with MW <3500) were identified for standard I by consuming only 200 fmol of each protein. When searched against a Swissprot database, the average sequence coverage for the standard I, II, and E. coli's ribosomal proteins were 57, 34, and 15%, respectively.

Amino Acid Sequence↗

PubMed2006

Clustering of childhood cancer in the inner city of Tehran metropolitan area: a GIS-based analysis.

The aim of this study was both to map the childhood cancer incidence in the districts of Tehran metropolitan area and to explore possible clustering of cancer cases in the diverse environments of this area. All incidence cases of childhood cancers (age group under 15 years) belonging to the 22 districts of Tehran metropolis and occurring during the period of 1998 till 2002 were ascertained from three sources. Each case's place of residency was geo-referenced. The scan statistics cluster detecting technique was used to evaluate clustering of cases throughout Tehran. The overall incidence rate (IR) of childhood cancer was 176.3/1,000,000 children under 15 years of age. The lowest IR among both boys and girls was observed in district 22 (69.4/1,000,000) and the highest was observed in district 6 (242.09/1,000,000). The detection of clusters was performed for all cancer sites. All the cancer sites combined category showed clustering in the districts 7, 13, 8, 6, 3, 14, 12, 11, and 4. For this category, the clustering likelihood was marginally statistically significant (p-value=0.056), with an overall relative risk of 1.30. No statistically significant patterns of clustering were detected for other categories.

Adolescent↗

PubMed2004

Application of capillary electrophoresis/ electrospray ionization-mass spectrometry to subcellular proteomics of Escherichia coli ribosomal proteins.

We introduce capillary electrophoresis-mass spectrometry (CE-MS) as an efficient means for the on-line separation and identification of protein mixtures. It was found that while CE/electrospray ionization (ESI)-MS analysis of whole-cell lysate was too complicated for the one-dimensional CE-MS analysis, the technique was useful for the analysis of protein mixtures of moderate complexity (approximately 50 intact proteins). CE/ESI-MS was applied to the subcellular proteomics of ribosomal Escherichia coli. 55 out of the 56 ribosomal proteins were detected with ease by using only approximately 3.4 ng of ribosomal proteins. In addition, it was found that the mass accuracy of the conventional MS (such as quadrupole ion traps) was good enough to identify many post-translational modifications of the intact proteins by simply comparing their measured average molecular weight with the average molecular weight predicted from gene banks.

Electrophoresis, Capillary↗

PubMed2004

Capillary electrophoresis-electrospray ionization mass spectrometry of amino acids, peptides, and proteins.

Separation in capillary electrophoresis (CE) is based on the movement of charged compounds inside a background electrolyte under an applied potential. Because the mechanism of separation of CE differs from that of conventional high-performance liquid chromatography (HPLC), where separation is based on the analyte's hydrophobic properties, CE is often used as a complementary technique to HPLC. In addition, because CE is performed in narrow capillaries at atmospheric pressure, it is used as an alternative to HPLC, capable of handling small sample volumes while providing shorter analysis times with higher efficiency. For the analysis of amino acid, protein, and peptide mixtures in small volume samples such as in single cells, CE has rapidly evolved as a preferred separation technique. The combination of a high-efficiency separation technique, such as CE, with mass spectrometry (MS) detection provides a powerful system for the analysis of complex biological mixtures. In this chapter, a theoretical and practical approach to achieving high-performance CE-MS is discussed and the utility of CE-MS for the analysis of amino acids, peptides, and proteins is demonstrated.

Amino Acid Sequence↗

PubMed2003

CE/electrospray ionization-MS analysis of underivatized d/l-amino acids and several small neurotransmitters at attomole levels through the use of 18-crown-6-tetracarboxylic acid as a complexation reagent/background electrolyte.

A new capillary electrophoresis/mass spectrometry technique is introduced for attomole detection of primary amines (including several neurotransmitters), amino acids, and their d/l enantiomers in one run through the use of a complexation reagent while using only approximately 1 nL of sample. The technique uses underivatized amino acids in conjunction with an underivatized capillary, which significantly reduces cost and analysis time. It was found that when (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18-C-6-TCA, MW 440) was used as the background electrolyte/complexation reagent during the capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) analysis of underivatized amino acids, stable complexes were formed between the amino acids and the 18-C-6-TCA molecules. These complexes, which exhibited high ionization efficiencies, were detectable at attomole levels for most amino acids. The detection limits of the AA/18-C-6-TCA complexes were on the average more than 2 orders of magnitude lower than that of the free amino acids in solution. In addition to lower detection limits under CE/ESI-MS, a solution of 18-C-6-TCA in the concentration range of 5-30 mM provided high separation efficiency for mixtures of l-amino acids as well as mixtures of d/l-amino acids. By using a solution of 18-C-6-TCA as the background electrolyte in conjunction with an underivatized, 130-cm-long, 20-microm-i.d., 150-microm-o.d. fused-silica capillary and by monitoring the m/z range of the amino acid/18-C-6-TCA complexes (m/z 515-700), most of the standard amino acids and many of their enantiomers were separated and detected with high separation efficiency and high sensitivity (nanomolar concentration detection limits) in one run. The solutions of 18-C-6-TCA also worked well as the CE/ESI-MS BGE for low-level detection of several neurotransmitters and some of their d/l enantiomers as well as for the analysis of amino acids at endogenous levels in lysed red blood cells.

Amino Acids↗

PubMed2003

Capillary electrophoresis to mass spectrometry interface using a porous junction.

A new capillary electrophoresis interface to electrospray ionization mass spectrometry (CE/ESI-MS) is introduced in which the electrical connection to the CE capillary outlet/ESI electrode is achieved by transfer of small ions related to the background electrolyte (BGE) through a porous section near the CE capillary outlet. In this design, only a small section of the capillary wall is made porous. The porous section is created by first thinning a small section of the capillary wall by drilling a well into it and then etching the remaining thin wall porous. This design has two advantages over previous designs (in which the whole circumference of the capillary was made porous): first, the capillary interface is more robust because only a small section of it is made porous, and therefore, no liquid junction is needed to secure the porous section. The electrical connection is achieved simply by inserting the capillary outlet containing the porous junction into the existing ESI needle and filling the needle with the BGE. Second, the time required to make the fused silica porous is reduced from approximately 1 h to a few minutes. In addition, there is no dead volume associated with the porous design, and because the actual metal/liquid contact occurs outside of the CE capillary, bubble formation due to redox reactions of water at the electrode does not affect CE/ESI-MS performance. The performance of this interface is demonstrated by the analyses of peptide and protein mixtures.

Journal Article↗

PubMed2003

Analysis of underivatized amino acids and their D/L-enantiomers by sheathless capillary electrophoresis/electrospray ionization mass spectrometry.

Capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was applied to the analysis of underivatized amino acids and the separation of their D/L-enantiomers. Under full-scan mode, all standard protein amino acids were separated and detected at low-femtomole levels using a 130-cm-long, 20-microm-i.d., 150-microm-o.d. underivatized fused-silica capillary with 1 M formic acid as the background electrolyte. The CE/ESI-MS technique was also applied to the separation of L-arginine from L-canavanine (a close analogue of arginine where the terminal methylene linked to the guanidine group of arginine is replaced by an oxygen atom) in a complex mixture containing all standard protein amino acids. The utility of CE/ESI-MS in the analysis of real-world samples was demonstrated by the identification of two metabolic diseases (PKU and tyrosinemia) through blood analysis with minimal sample preparation. In addition, the on-line separation of 11 underivatized L-amino acids from their D-enantiomers was achieved by using a 30 mM solution of (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid as the background electrolyte.

Amino Acids↗

PubMed2002

Analysis of carbonic anhydrase in human red Blood cells using capillary electrophoresis/ electrospray ionization-mass spectrometry.

Capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was applied to the analysis of human red blood cells (RBCs) using the split-flow technique for interfacing CE to MS. By using a long (approximately125-cm) and narrow (approximately 15-microm-i.d.) capillary, the four major proteins of the RBC, which are hemoglobin (Hb, alpha- and beta-chains, 900 amol/chain), carbonic anhydrase I (CAI, approximately 7 amol/cell), and carbonic anhydrase II (CAII, approximately 0.8 amol/cell), were separated from each other and detected at low-attomole levels in one run and minimal sample preparation. Under these conditions, the detection limits for CAI and CAII in lysed RBCs were approximately 20 and approximately 44 amol, respectively. The approximately 20-amol detection limit of CAI was confirmed by the CE/ESI-MS analysis of three intact RBCs that had been drawn into the capillary under a microscope. A shorter capillary (approximately 55 cm long) provided faster analysis time but did not separate CAII from the beta-chain of hemoglobin, causing the CAII signal to be masked by the background chemical noise generated by the approximately 1,000 x molar excess of the beta-chain. Under this condition, the CAII detection limit increased to approximately 500 amol. From three methods of sample introduction (injection of lysed blood, injection of intact cells under microscope, and injection of intact cells suspended in saline solution), injection of lysed blood provided the optimum sensitivity. It was found that a background electrolyte (BGE) containing 0.1% acetic acid in water worked best for the analysis of intact cells, while a BGE containing 0.1% acetic acid in water + acetonitrile (50/50 by volume) worked best for the analysis of lysed blood.

Carbonic Anhydrase I↗

PubMed2002

Capillary electrophoresis mass spectrometry and its application to the analysis of biological mixtures.

Capillary electrophoresis (CE) mass spectrometry (MS), with its ability to separate compounds present in extremely small volume samples rapidly, with high separation efficiency, and with compound identification capability based on molecular weight, is an extremely valuable analytical technique for the analysis of complex biological mixtures. The highest sensitivities and separation efficiencies are usually achieved by using narrow capillaries (5-50 micro m i.d.) and by using sheathless CE-to-MS interfaces. The difficulties in CE-to-MS interfacing and the limited loadability of these narrow columns, however, have prevented CE-MS from becoming a widely used analytical technique. To remedy these limitations, several CE-MS interfacing techniques have recently been introduced. While electrospray ionization is the most commonly used ionization technique for interfacing CE-to-MS, matrix assisted laser desorption ionization has also been used, using both on-line and off-line techniques. Moreover, the high concentration detection limit of CE has been addressed by development of several sample concentration and sample focusing methods. In addition, a wide variety of techniques such as capillary zone electrophoresis, capillary isoelectric focusing, and on-column transient isotachophoresis have now been interfaced to MS. These advances have resulted in a rapid increase in the use of CE-MS in the analysis of complex biological mixtures. CE-MS has now been successfully applied to the analysis of a wide variety of compounds including amino acids, protein digests, protein mixtures, single cells, oligonucleotides, and various small molecules relevant to the pharmaceutical industry.

Biology↗