Direct Quantitative Analysis of Biomarkers using Mass Spectrometry

Point-of-care (POC) diagnostics describes a step in the medical treatment process where drugs can be monitored in a patient’s body on-site and in a timely fashion. Mass spectrometry (MS) can provide a quick, efficient, and highly accurate method of analysis of patient biofluids and tissues. Developing methods to bring this diagnostic mechanism to hospitals and clinics has the potential to improve patient care through, for example, personalized medicine. Our goal was to develop a way to effectively introduce internal standard (IS), a necessary chemical for the analytical process, to low-volume biofluid samples. Additionally, the effective direct quantitation of biomarkers with MS was demonstrated using a rat model of nicotine metabolism and the detection of 3-HPMA in urine. By pre-coating silica glass capillary tubes with a fixed volume with IS, biological samples, such as blood, can be obtained in the tube through capillary action and mixed with the IS before deposition for analysis. This method was applied to several different drugs and they were analyzed using a triple quadrupole mass spectrometer. It was optimized for the detection of the metabolite cotinine through a study of solvents and elution processes. Additionally, cotinine was quantified in rat’s blood using this method and the acrolein metabolite 3-HPMA was quantified in urine. Additional work is needed to expand this method for the rapid detection of other biomarkers. In the future, this can contribute to the expanded use of MS in clinical care and improved POC diagnostics

Electron Transfer Dissociation of Amide Nitrogen Methylated Polypeptide Cations

Unmodified and amide nitrogen methylated peptide cations were reacted with azobenzene radical anions to study the utility of electron transfer dissociation (ETD) in analyzing N-methylated peptides. We show that methylation of the amide nitrogen has no deleterious effects on the ETD process. As a result, location of alkylation on amide nitrogens should be straightforward. Such a modification might be expected to affect the ETD process if hydrogen bonding involving the amide hydrogen is important for the ETD mechanism. The partitioning of the ion/ion reaction products into all of the various reaction channels was determined and compared for modified and unmodified peptide cations. While subtle differences in the relative abundances of the various ETD channels were observed, there is no strong evidence that hydrogen bonding involving the amide nitrogen plays an important role in the ETD process

Reactions of poly(ethylene glycol) cations with iodide and perfluorocarbon anions

AbstractMultiply charged poly(ethylene glycol) ions of the form (M+nNa)n+ derived from electrospray ionization have been subjected to reactions with negative ions in the quadrupole ion trap. Mixtures of multiply charged positive ions ranging in average mass from about 2000 to about 14,000 Da were observed to react with perfluorocarbon anions by either proton transfer or fluoride transfer. Iodide anions reacted with the same positive ions by attachment. In no case was fragmentation of the polymer ion observed. In all cases, the multiply charged positive ion charge states could be readily reduced to +1, thereby eliminating the charge state overlap observed in the normal electrospray mass spectrum. With all three reaction mechanisms, however, the +1 product ions were comprised of mixtures of products with varying numbers of sodium ions, and in the case of iodide attachment and fluoride transfer, varying numbers of halogen anions. These reactions shift the mass distributions to higher masses and broaden the distributions. The extents to which these effects occur are functions of the magnitudes of the initial charges and the width of the initial charge state distributions. Care must be taken in deriving information about the polymer molecular weight distribution from the singly charged product ions arising from these ion/ion reactions. The cluster ions containing iodide were shown to be intermediates in sodium ion transfer. Dissociation of the adduct ions can therefore lead to a +1 product ion population that is comprised predominantly of M+Na+ ions. However, a strategy based on the dissociation of the iodide cluster ions is limited by difficulties in dissociating high mass-to-charge ions in the quadrupole ion trap

Selective Ion Isolation/Rejection Over a Broad Mass Range in the Quadrupole Ion Trap

AbstractTechniques are presented for mass-selective ion manipulation over a wide mass range in a three-dimensional quadrupole. The methods use an auxiliary, low-amplitude radio-frequency signal applied to the endcap electrodes. This signal is either held at a single frequency as the fundamental radio-frequency trapping amplitude is ramped or swept over a frequency range while the fundamental radio-frequency trapping amplitude is held at a fixed level. Ion isolation and ejection are demonstrated for ions formed within the ion trap using electron ionization and for ions injected into the ion trap formed either by an air-sustained glow discharge or by electrospray. Mass-selective ion ejection is used to reduce matrix-ion-induced space charge during ion injection, thereby producing signal enhancement for the detection of 2,4,6-trinitrotoluene in air. Mass-selective isolation of ions with mass-to-charge ratios above the normal operating range (m/z 650) for the ion trap is also demonstrated after injection of myoglobin ions formed via electrospray

Ion-ion proton transfer reactions of bio-ions involving noncovalent interactions: Holomyoglobin

Multiply protonated horse skeletal muscle holomyoglobin and apomyoglobin have been subjected to ion-ion proton transfer reactions with anions derived from perfluoro-1,3dimethylcyclohexane in a quadrupole ion trap operated with helium as a bath gas at 1 mtorr. Neither the apomyoglobin nor holomyoglobin ions show any sign of fragmentation associated with charge state reduction to the 1 + charge state. This is particularly noteworthy for the holomyoglobin ions, which retain the noncovalently bound heme group. For example, no sign of heme loss is associated with charge state reduction from the 9 + charge state of holomyoglobin to the 1 + charge state despite the eight consecutive highly exothermic proton transfer reactions required to bring about this charge change. This result is consistent with calculations that show the combination of long ion lifetime and the high ion-helium collision rate relative to the ion-ion collision rate makes fragmentation unlikely for high mass ions in the ion trap environment even for noncovalently bound complexes of moderate binding strength. The ion-ion proton transfer rates for holo- and apomyoglobin ions of the same charge state also were observed to be indistinguishable, which supports the expectation that ion-ion proton transfer rates are insensitive to ion structure and are determined primarily by the attractive Coulomb field

Metabolic Comparison of Wild-Type and Transgenic Synechocystis PCC 6803 Cyanobacteria

The Calvin-Benson (CBB) cycle is an essential part of nature. This phenomenon allows carbon molecules in carbon dioxide from the atmosphere to be converted into useful energy in the form of sugars. Cyanobacteria are single-celled organisms capable of utilizing energy from sunlight to drive this cycle and are also readily engineered. In hopes of improving this cycle, we compared a wild-type version of the Synechocystis PCC6803 cyanobacteria to an engineered version overexpressing the enzyme FBA (fructose-biphosphate aldolase), called 70 glpX, to deduce how the overexpressing strain is able to be more photosynthetically efficient. To do this, comparative metabolomics were done to compare metabolite concentrations in order to identify differences between the two. It was found that the FBA enzyme in the 70 glpX contained increased metabolite concentrations at certain points in the CBB cycle when compared to the wild-type, causing an increase in the rate of photosynthesis. We can see that the substrate was higher at certain points, which may suggest a higher metabolic rate, explaining how the engineered version is better at carrying out photosynthesis

Probing the mechanisms of electron capture dissociation mass spectrometry with nitrated peptides

Previously we have shown that the presence of 3-nitrotyrosine within a peptide sequence severely depletes the peptide backbone fragments typically observed following electron capture dissociation (ECD) mass spectrometry. Instead, ECD of nitrated peptides is characterised by abundant losses of small neutrals (hydroxyl radicals, water and ammonia). Here, we investigate the origin of ammonia loss by comparing the ECD behaviour of lysine- and arginine-containing nitrated peptides, and their N-acetylated counterparts, and nitrated peptides containing no basic amino acid residues. The results reveal that ammonia loss derives from the N-terminus of the peptides, however, the key finding of this work is the insight provided into the hierarchy of various proposed ECD mechanisms: the Utah-Washington mechanism, the electron predator mechanism and the Oslo mechanism

Sodium-tolerant matrix for matrix-assisted laser desorption/ionization mass spectrometry and post-source decay of oligonucleotides

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