Liquid Chromatography Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometric Characterization of <i>N</i>-Linked Glycans and Glycopeptides

We combine liquid chromatography, electrospray ionization, and Fourier transform ion cyclotron resonance mass spectrometry (LC ESI FT-ICR MS) to determine the sugar composition, linkage pattern, and attachment sites of N-linked glycans. N-linked glycans were enzymatically released from glycoproteins with peptide N-glycosidase F, followed by purification with graphitized carbon cartridge solid-phase extraction and separation over a TSK-Gel Amide80 column under hydrophilic interaction chromatography (HILIC) conditions. Unique glycopeptide compositions were determined from experimentally measured masses for different combinations of glycans and glycopeptides. The method was validated by identifying four peptides glycosylated so as to yield 12 glycopeptides unique in glycan composition for the standard glycoprotein, bovine alpha-2-HS-glycoprotein. We then assigned a total of 137 unique glycopeptide compositions from 18 glycoproteins from fetal bovine serum, and the glycan structures for most of the assigned glycopeptides were heterogeneous. Highly accurate FT-ICR mass measurement is essential for reliable identification

Polar Lipid Composition of Biodiesel Algae Candidates <i>Nannochloropsis oculata</i> and <i>Haematococcus pluvialis</i> from Nano Liquid Chromatography Coupled with Negative Electrospray Ionization 14.5 T Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Algae lipids contain long-chain saturated and polyunsaturated fatty acids. The lipids may be transesterified to generate biodiesel fuel. Here, we compare polar lipid compositions for two microalgae, <i>Nannochloropsis oculata</i> and <i>Haematococcus pluvialis</i>, that are prospective lipid-rich feedstock candidates for an emerging biodiesel industry. Online nano liquid chromatography coupled with negative electrospray ionization 14.5 T Fourier transform ion cyclotron resonance mass spectrometry ((−)­ESI FT-ICR MS) with newly modified ion optics provides ultrahigh mass accuracy and resolving power to identify hundreds of unique elemental compositions. Assignments are confirmed by isotopic fine structure for a polar lipid extract. Collision-induced-dissociation (CID) MS/MS provides additional structural information. <i>H. pluvialis</i> exhibits more highly polyunsaturated lipids than does <i>N. oculata</i>

Unequivocal Determination of Site-Specific Protein Disulfide Bond Reduction Potentials by Top-Down FTICR MS: Characterization of the N- and C‑Terminal Redox-Active Sites in Human Thioredoxin 1

We report the reliable determination of equilibrium protein disulfide bond reduction potentials (<i>E</i>°′) by isotope-coded cysteine alkylation coupled with top-down Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). This technique enables multiple redox-active sites to be characterized simultaneously and unambiguously without the need for proteolysis or site-directed mutagenesis. Our model system was <i>E. coli</i> thioredoxin, and we determined <i>E</i>°′ for its CGPC active-site disulfide as −280 mV in accord with literature values. <i>E</i>°′ for the homologous disulfide in human thioredoxin 1 (Trx1) was determined as −281 mV, a value considerably more negative than the previously reported −230 mV. We also observed <i>S</i>-glutathionylation of Trx1 and localized that redox modification to Cys72; <i>E</i>°′ for the intermolecular disulfide was determined as −186 mV. Intriguingly, that value corresponds to the intracellular glutathione/glutathione disulfide (GSH/GSSG) potential at the redox boundary between cellular differentiation and apoptosis

Exact Masses and Chemical Formulas of Individual Suwannee River Fulvic Acids from Ultrahigh Resolution Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectra

Molecular formulas have been assigned for 4626 individual Suwannee River fulvic acids based on accurate mass measurements from ions generated by electrospray ionization and observed by ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). Formula assignments were possible because of the mass accuracy of FTICR MS at high field (9.4 T) and the regular mass spacing patterns found in fulvic acid mixtures. Sorting the 4626 individually observed ions according to Kendrick mass defect and nominal mass series (z* score) revealed that all could be assigned to 1 of 266 distinct homologous series that differ in oxygen content and double bond equivalence. Tandem mass spectrometry based on infrared multiphoton dissociation identified labile fragments of fulvic acid molecules, whose chemical formulas led to plausible structures consistent with degraded lignin as a source of Suwannee River fulvic acids

Use of Saturates/Aromatics/Resins/Asphaltenes (SARA) Fractionation To Determine Matrix Effects in Crude Oil Analysis by Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

We have previously demonstrated the ability of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) to resolve and identify the polar species found in all petroleum distillates. The ultrahigh resolving power and mass accuracy of FT-ICR MS allow for the identification of thousands of compounds in crude oils without prior chromatographic separation. Here, we compare positive-ion ESI FT-ICR mass spectra of a South American crude oil with spectra of its saturates/aromatics/resins/asphaltenes (SARA)-isolated asphaltenes, resins, and aromatics, to ascertain the effect of the other components on the relative mass spectral abundances of the polar aromatics. Saturates are unobservable by ESI. For the chosen oil, little to no signal was obtained for the asphaltenes and resins because of their mostly acidic nature. The mass distributions, heteroatom “class” distributions, “type” (rings plus double bonds) distributions, and carbon number distributions of the aromatic fraction and unfractionated crude oil were highly similar. Thus, the saturates, asphaltenes, and resins do not affect the relative abundances of polar aromatics observed by positive-ion electrospray FT-ICR MS. It is therefore not necessary to isolate the polar aromatic fraction to characterize its chemical composition in a petroleum crude oil

Use of Saturates/Aromatics/Resins/Asphaltenes (SARA) Fractionation To Determine Matrix Effects in Crude Oil Analysis by Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

We have previously demonstrated the ability of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) to resolve and identify the polar species found in all petroleum distillates. The ultrahigh resolving power and mass accuracy of FT-ICR MS allow for the identification of thousands of compounds in crude oils without prior chromatographic separation. Here, we compare positive-ion ESI FT-ICR mass spectra of a South American crude oil with spectra of its saturates/aromatics/resins/asphaltenes (SARA)-isolated asphaltenes, resins, and aromatics, to ascertain the effect of the other components on the relative mass spectral abundances of the polar aromatics. Saturates are unobservable by ESI. For the chosen oil, little to no signal was obtained for the asphaltenes and resins because of their mostly acidic nature. The mass distributions, heteroatom “class” distributions, “type” (rings plus double bonds) distributions, and carbon number distributions of the aromatic fraction and unfractionated crude oil were highly similar. Thus, the saturates, asphaltenes, and resins do not affect the relative abundances of polar aromatics observed by positive-ion electrospray FT-ICR MS. It is therefore not necessary to isolate the polar aromatic fraction to characterize its chemical composition in a petroleum crude oil

New Reagents for Enhanced Liquid Chromatographic Separation and Charging of Intact Protein Ions for Electrospray Ionization Mass Spectrometry

Electrospray ionization produces multiply charged ions, thereby lowering the mass-to-charge ratio for peptides and small proteins to a range readily accessed by quadrupole ion trap, orbitrap, and ion cyclotron resonance (ICR) mass analyzers (m/z = 400−2000). For Fourier transform mass analyzers (orbitrap and ICR), higher charge also improves signal-to-noise ratio, mass resolution, and mass accuracy. Addition of m-nitrobenzyl alcohol (m-NBA) or sulfolane has previously been shown to increase the charge states of proteins. Moreover, polar aprotic dimethylformamide (DMF) improves chromatographic separation of proteolytic peptides for mass analysis of solution-phase protein hydrogen/deuterium exchange for improved (78−96%) sequence coverage. Here, we show that addition of each of the various modifiers (DMF, thiodiglycol, dimethylacetamide, dimethylsulfoxide, and N-methylpyrrolidone) can significantly increase the charge states of proteins up to 78 kDa. Moreover, incorporation of the same modifiers into reversed-phase liquid chromatography solvents improves sensitivity, charging, and chromatographic resolution for intact proteins

Use of Saturates/Aromatics/Resins/Asphaltenes (SARA) Fractionation To Determine Matrix Effects in Crude Oil Analysis by Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

We have previously demonstrated the ability of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) to resolve and identify the polar species found in all petroleum distillates. The ultrahigh resolving power and mass accuracy of FT-ICR MS allow for the identification of thousands of compounds in crude oils without prior chromatographic separation. Here, we compare positive-ion ESI FT-ICR mass spectra of a South American crude oil with spectra of its saturates/aromatics/resins/asphaltenes (SARA)-isolated asphaltenes, resins, and aromatics, to ascertain the effect of the other components on the relative mass spectral abundances of the polar aromatics. Saturates are unobservable by ESI. For the chosen oil, little to no signal was obtained for the asphaltenes and resins because of their mostly acidic nature. The mass distributions, heteroatom “class” distributions, “type” (rings plus double bonds) distributions, and carbon number distributions of the aromatic fraction and unfractionated crude oil were highly similar. Thus, the saturates, asphaltenes, and resins do not affect the relative abundances of polar aromatics observed by positive-ion electrospray FT-ICR MS. It is therefore not necessary to isolate the polar aromatic fraction to characterize its chemical composition in a petroleum crude oil

Use of Saturates/Aromatics/Resins/Asphaltenes (SARA) Fractionation To Determine Matrix Effects in Crude Oil Analysis by Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

We have previously demonstrated the ability of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) to resolve and identify the polar species found in all petroleum distillates. The ultrahigh resolving power and mass accuracy of FT-ICR MS allow for the identification of thousands of compounds in crude oils without prior chromatographic separation. Here, we compare positive-ion ESI FT-ICR mass spectra of a South American crude oil with spectra of its saturates/aromatics/resins/asphaltenes (SARA)-isolated asphaltenes, resins, and aromatics, to ascertain the effect of the other components on the relative mass spectral abundances of the polar aromatics. Saturates are unobservable by ESI. For the chosen oil, little to no signal was obtained for the asphaltenes and resins because of their mostly acidic nature. The mass distributions, heteroatom “class” distributions, “type” (rings plus double bonds) distributions, and carbon number distributions of the aromatic fraction and unfractionated crude oil were highly similar. Thus, the saturates, asphaltenes, and resins do not affect the relative abundances of polar aromatics observed by positive-ion electrospray FT-ICR MS. It is therefore not necessary to isolate the polar aromatic fraction to characterize its chemical composition in a petroleum crude oil

Use of Saturates/Aromatics/Resins/Asphaltenes (SARA) Fractionation To Determine Matrix Effects in Crude Oil Analysis by Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

We have previously demonstrated the ability of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) to resolve and identify the polar species found in all petroleum distillates. The ultrahigh resolving power and mass accuracy of FT-ICR MS allow for the identification of thousands of compounds in crude oils without prior chromatographic separation. Here, we compare positive-ion ESI FT-ICR mass spectra of a South American crude oil with spectra of its saturates/aromatics/resins/asphaltenes (SARA)-isolated asphaltenes, resins, and aromatics, to ascertain the effect of the other components on the relative mass spectral abundances of the polar aromatics. Saturates are unobservable by ESI. For the chosen oil, little to no signal was obtained for the asphaltenes and resins because of their mostly acidic nature. The mass distributions, heteroatom “class” distributions, “type” (rings plus double bonds) distributions, and carbon number distributions of the aromatic fraction and unfractionated crude oil were highly similar. Thus, the saturates, asphaltenes, and resins do not affect the relative abundances of polar aromatics observed by positive-ion electrospray FT-ICR MS. It is therefore not necessary to isolate the polar aromatic fraction to characterize its chemical composition in a petroleum crude oil