Ionization Triple Quadrupole Mass Spectrometer Research Articles

Validated HPLC–MS/MS method for simultaneous determination of simvastatin and simvastatin hydroxy acid in human plasma

Cholesterol lowering statin drugs are the most frequently prescribed agents for reducing morbidity and mortality related to coronary heart disease. This publication presents a validated, highly sensitive and selective isocratic HPLC method for the quantitative determination of the major statin drug simvastatin (SIM) and its metabolite simvastatin hydroxy acid (SIMA). Detection was performed on an electrospray ionization triple quadrupole mass spectrometer equipped with an ESI interface operated in positive and negative ionization mode. The multiple reaction-monitoring mode (MRM) was used to provide MS/MS detection. The linearity for the calibration curve in the concentration range of 0.10–16.00 ng/mL for SIM and 0.10–16.00 ng/mL for SIMA is presented. Inter- and intra-day precision and accuracy of the proposed method were characterized by relative standard deviation (R.S.D.) and percentage deviation, respectively; with both lower than 7% for all analytes. The limit of quantitation was 0.03 ng/mL for SIM and 0.02 ng/mL for SIMA. The devised method was employed in the pharmacokinetic study of SIM and the pharmacokinetic parameters of all analytes are also presented.

Synthesis of 5-fluoro-β-D-glucopyranosyluronic acid fluoride and its evaluation as a mechanistic probe of Escherichia coli β-glucuronidase

Synthesis of the potential mechanism-based inactivator of β-D-glucuronidases (5-fluoro-β-D-glucopyranosyluronic acid fluoride) was accomplished via a six-step process from D-glucuronic acid that involved radical bromination at C-5 and displacement of the bromide by fluoride. A key step in this process was the masking of the carboxylic acid as a phenacyl ester. This group is uniquely stable to conditions of photobromination and fluoride displacement, yet removable under very mild conditions. Incubation of the Escherichia coli β-glucuronidase with 5-fluoro-β-D-glucopyranosyluronic acid fluoride resulted in time-dependent inactivation of the enzyme through the accumulation of a covalent 5-fluoro-α-D-glucopyranosyluronic acid-enzyme. Peptic digestion of the 5-fluoro-α-D-glucopyranosyluronic acid-enzyme intermediate and subsequent analysis by liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer indicated the presence of a 5-fluoro-α-D-glucopyranosyluronic acid-modified peptide. This peptide was partially purified by HPLC and its sequence determined by tandem mass spectrometry in the daughter ion scan mode, permitting the identification of Glu504 as the catalytic nucleophile within the sequence ITEYGVD. This new reagent is therefore useful for the specific, mechanism-based inactivation of glycuronidases and has good potential in other studies of enzymes of this general class.Key words: β-glucuronidase, catalytic nucleophile, 5-fluoro-β-D-glucopyranosyluronic acid fluoride, electrospray MS.

Mass spectral characterization of a protein-nucleic acid photocrosslink.

A photocrosslink between basic fibroblast growth factor (bFGF155) and a high affinity ssDNA oligonucleotide was characterized by positive ion electrospray ionization mass spectrometry (ESIMS). The DNA was a 61-mer oligonucleotide photoaptamer bearing seven bromodeoxyuridines, identified by in vitro selection. Specific photocrosslinking of the protein to the oligonucleotide was achieved by 308 nm XeCl excimer laser excitation. The cross-linked protein nucleic acid complex was proteolyzed with trypsin. The resulting peptide crosslink was purified by PAGE, eluted, and digested by snake venom phosphodiesterase/alkaline phosphatase. Comparison of the oligonucleotide vs. the degraded peptide crosslink by high performance liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer showed a single ion unique to the crosslinked material. Sequencing by collision induced dissociation (MS/MS) on a triple quadrupole mass spectrometer revealed that this ion was the nonapeptide TGQYKLGSK (residues 130-138) crosslinked to a dinucleotide at Tyr133. The MS/MS spectrum indicated sequential fragmentation of the oligonucleotide to uracil covalently attached to the nonapeptide followed by fragmentation of the peptide bonds. Tyr133 is located within the heparin binding pocket, suggesting that the in vitro selection targeted this negative ion binding region of bFGF155.

Identification of the Active Site Nucleophile in Jack Bean α-Mannosidase Using 5-Fluoro-β-l-Gulosyl Fluoride

Mannosidases play a key role in the processing of glycoproteins and thus are of considerable pharmaceutical interest and indeed have emerged as targets for the development of anti-cancer therapies. Access to useful quantities of the mammalian enzymes has not yet been achieved; therefore, jack bean mannosidase, a readily available enzyme, has become the model system. However, the relevance of this enzyme has not been demonstrated, nor is anything known about the active site structure of this, or any other, mannosidase. Hydrolysis by this enzyme occurs with net retention of sugar anomeric configuration; thus, a double displacement mechanism involving a mannosyl-enzyme intermediate is presumably involved. Two new mechanism-based inhibitors, 5-fluoro-alpha-D-mannosyl fluoride and 5-fluoro-beta-L-gulosyl fluoride, which function by the steady state trapping of such an intermediate, have been synthesized and tested. Both show high affinity for jack bean alpha-mannosidase (Ki' = 71 and 86 microM, respectively), and the latter has been used to label the active site nucleophile. The labeled peptide present in a peptic digest of this trapped glycosyl-enzyme intermediate was identified by neutral loss scans on an electrospray ionization triple quadrupole mass spectrometer. Comparative liquid chromatographic/mass spectrometric analysis of peptic digests of labeled and unlabeled enzyme samples confirmed the unique presence of this peptide of m/z 1180.5 in the labeled sample. The label was cleaved from the peptide by treatment with ammonia, and the resultant unlabeled peptide was purified and sequenced by Edman degradation. The peptide identified contained only one candidate for the catalytic nucleophile, an aspartic acid. This residue was contained within the sequence Gly-Trp-Gln-Ile-Asp-Pro-Phe-Gly-His-Ser, which showed excellent sequence similarity with regions in mammalian lysosomal and Golgi alpha-mannosidase sequences. These mammalian alpha-mannosidases belong to family 38 (or class II alpha-mannosidases) in which the Asp in the above sequence is totally conserved. This finding therefore assigns jack bean alpha-mannosidase to family 38, validating it as a model for other pharmaceutically interesting enzymes and thereby identifying the catalytic nucleophile within this family.

Assignment of Sweet Almond β-Glucosidase as a Family 1 Glycosidase and Identification of Its Active Site Nucleophile

Sweet almond beta-glucosidase is a well studied glycosidase, having been subjected to numerous kinetic analyses and inhibition studies. However, it is not known to which glycosidase family it belongs, nor is the identity of the active site nucleophile known with certainty. It can be inactivated using the specific, mechanism-based enzyme inactivator 2-deoxy-2-fluoro-beta-D-glucopyranosyl fluoride, which functions by forming a stable 2-deoxy-2-fluoro-alpha-D-glucopyranosyl-enzyme intermediate. The glycosylated peptide present in a peptic digest of this trapped glycosyl-enzyme intermediate was identified by use of neutral loss scans on an electrospray ionization triple quadrupole mass spectrometer. Comparative liquid chromatographic/mass spectrometric analysis of peptic digests of labeled and unlabeled enzyme samples confirmed the unique presence of this peptide of m/z = 1041 in the labeled sample. The sequence of this peptide was determined to be Ile-Thr-Glu-Gln-Gly-Val-Asp-Glu by further tandem mass spectrometric analysis in the daughter ion scan mode in conjunction with Edman degradation of the purified peptide. The identity of the labeled side chain was determined by further tandem mass spectrometric analysis in the daughter ion scan mode of a partially purified sample of the labeled peptide subjected to methyl esterification, the fragmentation pattern being consistent only with the first Glu in the sequence being labeled. The sequence around this residue is identical to that surrounding the catalytic nucleophile in many members of glycosidase Family 1, confirming the assignment of this enzyme to that family. The residue labeled is, however, different from that (Asp) identified previously in the enzyme from bitter almonds by use of conduritol epoxide affinity labels, although apparently close in the primary sequence.

High sensitivity identification of proteins by electrospray ionization tandem mass spectrometry: initial comparison between an ion trap mass spectrometer and a triple quadrupole mass spectrometer.

Recently, we have shown that a solid-phase-microextraction/capillary electrophoresis device coupled to an electrospray ionization triple quadrupole mass spectrometer through a microelectrospray interface represents a powerful analytical system for the rapid, conclusive and sensitive identification of proteins separated by gel electrophoresis. Here we report on the successful coupling of the same device to an electrospray ionization ion trap mass spectrometer and on the comparative evaluation of the performance of the triple quadrupole and ion-trap-based systems. In the ion trap mass spectrometer-based system, using a tryptic digest of a calibrated bovine serum albumin sample, we achieved limits of detection in the single mass spectrometry (MS) and tandem MS mode, respectively, of 400 amol (if 20 microL of solution at a concentration of 20 amol/microL was applied). The system was also successfully used to identify six yeast proteins isolated from a single analytical two-dimensional polyacrylamide gel. For the detection of unfragmented peptide ions both systems showed comparable sensitivity, whereas the ion-trap-based system showed superior performance with fragment ion spectra.

Energy resolved mass spectrometry of diethyl alkyl phosphonates with an atmospheric pressure ionization tandem mass spectrometer

AbstractEnergy resolved mass spectrometry (ERMS) was used to characterize the dissociation processes of four diethyl alkyl phosphonates where the alkyl R group attached to the central phosphorus atom ranged from methyl to n‐butyl. An atmospheric pressure ionization triple quadrupole mass spectrometer was used in the analyses. Dissociation of the MH+ and further dissociation of the resulting progeny ions was induced between the atmospheric—vacuum sampling orifice and the first ion focusing RF‐only quadrupole, Q0, instead of the central Q2 quadrupole. The major dissociation pathways of the four phosphonates could be characterized by two sequential McLafferty rearrangements yielding an alkyl phosphonic acid. This acid further dissociated losing H2O followed by ROH to produce PO+, or the same process occurred but in the reverse order, i.e. first losing ROH followed by H2O to yield PO+. The latter process was the energetically favored process. The alkyl group attached to the phosphorus atom of the protonated alkyl phosphonic acid was observed to have a significant influence on the stability of the protonated alkyl phosphonic acid. As the R group increased in size with a concomitant increase in electron‐donating power, the alkyl phosphonic acid was less prone to dissociation than other alkyl phosphonic acids which possessed smaller R groups. A significant difference was also noted in the alkyl phosphonic acid dissociation processes; methyl phosphonic acid dissociated predominantly via H2O loss unlike the other acids which favored the loss of ROH over H2O.