Mehdi Moini

Sodium trifluoroacetate as a tune/calibration compound for positive- and negative-ion electrospray ionization mass spectrometry in the mass range of 100–4000 Da

We have identified aqueous:acetonitrile solutions of alkali-metal trifluoroacetate compounds as tune/calibration standards for both positive- and negative-ion electrospray ionization mass spectrometry (ESI/MS). Each alkali-metal trifluoroacetate solution in water and acetonitrile (50:50, v/v) yields evenly spaced, singly charged peaks in the mass range of 100–3500 Da. Intense peaks are formed either by infusing the solution using a syringe pump, by infusing the solution into a stream of liquids [such as a high-performance liquid chromatography (HPLC) mobile phase] flowing to the electrospray needle, or by injecting the salt-containing solution into an HPLC mobile phase containing trifluoroacetic acid. The advantages of these compounds include: (i) generation of singly charged ions in both positive and negative ionization modes in the mass range of approximately 100–3500 Da, (ii) formation of evenly spaced peaks with similar intensity across the entire mass range, (iii) the most abundant isotope in each mass cluster is the lowest mass peak (monoisotopic mass), which is free from variation in natural isotope distribution, (iv) commercial availability, (v) they easily dissolve in common liquid chromatography solvents, and (vi) lack of any long-lasting memory effects or background problems.

Analysis of peptides, proteins, protein digests, and whole human blood by capillary electrophoresis/electrospray ionization-mass spectrometry using an in-capillary electrode sheathless interface

An in-capillary electrode sheathless interface was applied to the capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) analysis of mixtures of small peptides, proteins, and tryptic digests of proteins. The effects of different experimental parameters on the performance of this CE/ESI-MS interface were studied. The distance of the in-capillary electrode from the CE outlet and the length of the electrode inside the capillary had no significant effects on the CE separation and ESI behavior under the experimental conditions used. However, significant enhancement of the sensitivity resulted from the use of narrower CE capillaries. Using a quadrupole mass spectrometer, an aminopropylsilane-coated capillary, and a wide scan mass-to-charge ratio range of 500–1400, detection limits of approximately 4, 1, and 0.6 fmol for cytochrome c and myoglobin were achieved for 75-, 50-, and 30-µm inner diameter capillaries, respectively. Approximately one order of magnitude lower detection limits were achieved under the multiple-ion monitoring mode. The application of the in-capillary electrode sheathless interface to real-world samples was demonstrated by CE/ESI-MS analysis of a human blood sample.

Identification of nonprotein amino acids from cycad seeds as N-ethoxycarbonyl ethyl ester derivatives by positive chemical-ionization gas chromatography-mass spectrometry

Nonprotein amino acids from nine species of cycad seeds were analyzed as N-ethoxycarbonyl ethyl ester (ECEE) derivatives by positive chemical-ionization gas chromatography-mass spectrometry. Based on the retention times and mass spectrometry analyses, 12 nonprotein amino acids were identified in these seeds. In addition to the excitatory and putative neurotoxin beta-N-methylamino-L-alanine (BMAA), the known neurotoxin beta-N-oxalylamino-L-alanine (BOAA) was detected from the seeds of Macrozamia moorei and M. communis, and delta-N-oxalyl-ornithine was obtained from the Cycas revoluta seeds. A novel nonprotein amino acid named cycasindene, previously reported from C. revoluta, was also found in the seeds of members of the C. angulata and C. rumphii complex. Eight additional known nonprotein amino acids were also identified. This is the first report of the neurotoxin BOAA from cycad seeds.

Self-Assembly of Pyrrole–Ferrocene Hybrids, Determined Inter Alia by a New Chemically Induced Electrospray Mass Spectrometry Technique

An interesting example of a noncovalently linked organometallic superstructure (shown right) is obtained from the self-assembly of tetrapyrrole-substituted ferrocene. As a result of the self-associating properties of the pyrrole–ester subunits, this system forms ribbon-like infinite chains in the solid state and small but well-characterized oligomers in organic solvents. Selective oxidation of the ferrocene moiety provides a cationic ferrocenium species amenable to electrospray mass spectrometric analysis.

Analysis of underivatized amino acid mixtures using high performance liquid chromatography/dual oscillating nebulizer atmospheric pressure microwave induced plasma ionization-mass spectrometry

A dual oscillating capillary nebulizer (OCN) in conjunction with an atmospheric pressure microwave induced plasma ionization (AP-MIPI) source was applied to the analysis of underivatized amino acid mixtures. It was found that, compared to the single OCN, the dual OCN enhanced the sensitivity of detection several fold. Enhanced sensitivity was compound dependent. For small molecules, such as amino acids, it was 2–5 times more sensitive, while for larger molecules such as peptides it was more than an order of magnitude. The increase in sensitivity was attributed to the enhanced nebulization of the new torch. By using water/ acetonitrile containing 0.1% nonafluoropentanoic acid as the high performance liquid chromatography (HPLC) mobile phase and a C18 column, all common amino acids were separated and detected. A comparison between the results obtained using microwave induced plasma, atmospheric pressure chemical ionization (APCI), and electrospray ionization (ESI) at flow rates compatible with micro LC (10–100 μL/min) showed a higher sensitivity of detection with the AP-MIPI technique for the analysis of underivatized amino acids.

CAPILLARY ELECTROPHORESIS/ELECTROSPRAY IONIZATION HIGH MASS ACCURACY TIME-OF-FLIGHT MASS SPECTROMETRY FOR PROTEIN IDENTIFICATION USING PEPTIDE MAPPING

Capillary electrophoresis/electrospray ionization (CE/ESI) high mass accuracy time-of-flight mass spectrometry was used for the first time to characterize small proteins using peptide mapping. To identify small proteins, the intact proteins were first analyzed to obtain their average molecular weights with errors less than 1 Da. On-line capillary electrophoresis mass spectrometry of the tryptic digests of these small proteins was then performed to obtain the accurate molecular weights of the peptides with accuracies of approximately 10 ppm. Next, this information was used for the identification of the proteins using a protein database. It was found that high mass accuracy is an effective tool in reducing the list of most-likely proteins generated by the database. In addition, on-line collision-induced dissociation of the completely or partially resolved capillary electrophoresis peaks of the protein digests was used to unambiguously identify the sequences of these peptides. Each CE/ESI-MS analysis used only 5 nL of sample containing approximately 120 fmol of each peptide in protein digests. The results indicate that the combination of capillary electrophoresis and high resolution, high mass accuracy time-of-flight mass spectrometry is a viable option for the identification of small proteins using peptide mapping.

Separation and detection of the α- and β-chains of hemoglobin of a single intact red blood cell using capillary electrophoresis/ electrospray ionization time-of-flight mass spectrometry

A single intact red blood cell (erythrocyte) was injected into a capillary electrophoresis column, and following in-capillary lysing the α- and β-chains of the hemoglobin (∼450 amol) were separated and detected using capillary electrophoresis/electrospray ionization time-of-flight mass spectrometry. The mass spectra of the electropherogram peaks of the α and β chains showed identifiable peaks corresponding to multiply protonated and sodiated α- and β-chains of hemoglobin.

Ultramark 1621 as a reference compound for positive and negative ion fast-atom bombardment high-resolution mass spectrometry

Ultramark 1621, a commercially available mixture of fluorinated phosphazines, was found to be a useful calibration compound for negative and positive ion fast-atom bombardment (FAB) high-resolution mass spectrometry. Ultramark 1621 worked very well with the most widely used matrices such as glycerol, nitrobenzyl alcohol, and triethanolamine. The negative and positive ion FAB mass spectra of Ultramark include a series of intense peaks extending from 700 to 1900 u.

Quantitative analysis of fluorinated ethylchloroformate derivatives of protein amino acids and hydrolysis products of small peptides using chemical ionization gas chromatography-mass spectrometry

Abstract The derivatization and extraction efficiencies of 16 protein amino acids were investigated using trifluoroethanol+ethylchloroformate+pyridine as the derivatization reagents and chloroform as the solvent. The derivatization efficiencies ranged from 90% to 99% for all the amino acids studied except aspartic acid (79%). The extraction efficiencies of the derivatized amino acids using chloroform were close to 100%. In addition, the detection limits and linear dynamic ranges of trifluoroethanol ethylchloroformate (TFE-ECF) derivatives of these amino acids were studied using positive-ion chemical ionization gas chromatography-mass spectrometry (GC-MS). The detection limits of the protein amino acids were mostly in the low femtomole range. The linear dynamic ranges of these amino acids were in the range of zero to three orders of magnitude. The GC-MS analysis of the derivatized amino acids was applied to the hydrolysis products of Equal, a sugar substitute containing aspartame and diprotin B, a tripeptide. The results demonstrate that the TFE-ECF derivatization of the hydrolysis products of small peptides followed by GC-MS analysis can be used for the identification of their amino acid composition (except arginine) and for their complete amino acid analysis.

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 analytes 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.