Discovery of a biomarker for ß-Thalassemia by HPLC-MS and improvement from Proton Transfer Reaction - Parallel Ion Parking.

ß-thalassemia is a quantitative hemoglobin (Hb) disorder resulting in reduced production of Hb A and increased levels of Hb A2. Diagnosis of ß-thalassemia can be problematic when combined with other structural Hb variants, so that the separation approaches in routine clinical centers are not sufficiently decisive to obtain accurate results. Here, we separate the intact Hb subunits by high-performance liquid chromatography, followed by top-down tandem mass spectrometry of intact subunits to distinguish Hb variants. Proton transfer reaction-parallel ion parking (PTR-PIP), in which a radical anion removes protons from multiply charged precursor ions and produces charge-reduced ions spanning a limited m/z range, was used to increase the signal-to-noise ratio of the subunits of interest. We demonstrate that the δ/ß ratio can act as a biomarker to identify ß-thalassemia in normal electrospray ionization MS1 and PTR-PIP MS1. The application of PTR-PIP significantly increases the sensitivity and specificity of the HPLC-MS method to identify δ/ß ratio as a thalassemia biomarker.

Analysis of Isotopically Depleted Proteins Derived from Escherichia coli and Caenorhabditis elegans Cell Lines by Liquid Chromatography 21 T Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry.

Protein mass measurement by mass spectrometry is complicated by wide isotopic distributions that result from incorporation of heavy isotopes of C, H, N, O, and S, thereby limiting signal-to-noise ratio (SNR) and accurate intact mass determination, particularly for larger proteins [Fenselau et al. Anal. Chem. 1983, 55 (2), 353-356]. Observation of the monoisotopic mass-to-charge ratio (m/z) is the simplest and most accurate way to determine intact protein mass, but as mass increases, the relative abundance of the monoisotopic peak becomes so low that it is often undetectable. Here, we used an isotopically depleted growth medium to culture bacterial cells (Escherichia coli), resulting in isotopically depleted proteins. Isotopically depleted proteins show increased sequence coverage, mass measurement accuracy, and increased S/N of the monoisotopic peak by Fourier transform ion cyclotron resonance mass spectrometry analysis. We then grew Caenorhabditis elegans cells in a medium containing living isotopically depleted E. coli cells, thereby producing the first isotopically depleted eukaryotic proteins. This is the first time isotopic depletion has been implemented for four isotopes (1H, 12C, 14N, and 16O), resulting in the highest degree of depletion ever used for protein analysis and further improving MS analysis.

Complex Mixture Analysis of Emerging Contaminants Generated from Coal Tar- and Petroleum-Derived Pavement Sealants: Molecular Compositions and Correlations with Toxicity Revealed by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Pavement sealants are of environmental concern because of their complex petroleum-based chemistry and potential toxicity. Specifically, coal tar-derived sealants contain high concentrations of toxic/carcinogenic polycyclic aromatic hydrocarbons (PAHs) that, when weathered, can be transferred into the surrounding environment. Previous studies have demonstrated the effects of coal tar sealants on PAH concentration in nearby waterways and their harmful effects in aquatic ecosystems. Here, we investigate and compare the molecular composition of two different pavement sealants, petroleum asphalt- and coal tar-derived, and their photoproducts, by positive-ion (+) atmospheric pressure photoionization (APPI) and negative-ion (-) electrospray ionization (ESI) coupled with ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry to address species (high-boiling and/or high oxygen content) that lie outside the analytical window of other techniques due to ultra-high molecular complexity. In addition, we evaluate the toxicity of the water-soluble photoproducts by use of Microtox bioassay. The results demonstrate that the coal tar sealant contains higher amounts of PAHs and produces abundant water-soluble compounds, relative to unweathered materials, with a high abundance of PAH-like molecules of high toxicity. By comparison, the asphalt sealant produces fewer toxic water-soluble species, with molecular compositions that are consistent with natural dissolved organic matter. These results capture the mass, chemical diversity, toxicity, and source/photoproduct relationship of these compositionally complex emerging contaminants from the built environment.

Characterization of Structural Hemoglobin Variants by Top-Down Mass Spectrometry and R Programming Tools for Rapid Identification.

Hemoglobinopathies are one of the most prevalent genetic disorders, affecting millions throughout the world. These are caused by pathogenic variants in genes that control the production of hemoglobin (Hb) subunits. As the number of known Hb variants has increased, it has become more challenging to obtain unambiguous results from routine chromatographic assays employed in the clinical laboratory. Top-down proteomic analysis of Hb by mass spectrometry is a definitive method to directly characterize the sequences of intact subunits. Here, we apply "chimeric ion loading" to characterize Hb ß subunit variants. In this technique, product ions derived from complementary dissociation techniques are accumulated in a multipole storage device before delivery to a 21 T Fourier-transform ion cyclotron resonance mass spectrometer for simultaneous detection. To further improve the efficiency of identification of Hb variants and localization of the mutation site(s), we developed an R programming script, "Variants Identifier", to search top-down data against a database containing accurate intact mass differences and diagnostic ions from investigated Hb variants. A second R script, "PredictDiag", was developed and employed to determine relevant diagnostic ions for additional Hb variants with known sequences. These two R scripts were successfully applied to the identification of a Hb δ-ß fusion protein and other Hb variants. The combination of chimeric ion loading and the above R scripts enables rapid and reliable interpretation of top-down mass spectrometry data, regardless of activation type, for Hb variant identification.

Role of Molecular Structure in the Production of Water-Soluble Species by Photo-oxidation of Petroleum.

Asphaltenes are high-boiling and recalcitrant compounds that are generally minor components of crude oil (∼0.1-15.0 wt %) but dominate the composition of heavily weathered spilled petroleum. These solid residues exhibit a high structural complexity, comprised of polycyclic aromatic hydrocarbons (PAHs) that are a mixture of single-core (island) and multicore (archipelago) structural motifs. The mass fraction of each motif is sample-dependent. Thus, knowledge of a potential structural dependence (single- versus multicore) on the production of water-soluble species from asphaltene samples is key to understanding the contribution of photochemically generated dissolved organic matter from oil spills. In this work, asphaltene samples with enriched mass fractions of either island (single-core) or archipelago (multicore) structural motifs are photo-oxidized on artificial seawater by the use of a solar simulator. Molecular characterization of oil- and water-soluble photoproducts, conducted by Fourier transform ion cyclotron resonance mass spectrometry, reveals that island motifs exhibit very limited production of water-soluble species, and their oil-soluble products reflect the molecular composition of the starting material. Conversely, archipelago motifs yield a water-soluble compositional continuum of Ox, SxOy, and NxOy containing hydrocarbons species that exhibit the typical molecular fingerprint of dissolved organic matter (DOM). The lower carbon number and aromaticity of the archipelago-derived asphaltene photoproducts suggest the occurrence of photofragmentation (or photolysis) reactions. To investigate the possibility of the opposite reaction (photopolymerization), the photo-oxidation of small PAHs isolated from a low-boiling petroleum distillation cut was also performed. It yielded water-soluble compounds with carbon number and aromaticity up to 2-fold higher than the starting material, strongly suggesting that polymerization (addition reactions) occurs. Collectively, the results indicate that the presence of archipelago motifs and the occurrence of cracking/polymerization reactions are central in the production of dissolved organic matter from fossil fuels.

Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry.

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.

Characterization of an Asphalt Binder and Photoproducts by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Reveals Abundant Water-Soluble Hydrocarbons.

Road asphalt is comprised of aggregate (rocks) mixed with a binder composed of high-boiling petroleum-derived compounds, which have been thought to be relatively inert (unreactive) and thus leach small amounts of polyaromatic hydrocarbons (PAHs) into water from the built environment. However, recent studies have demonstrated that petroleum readily undergoes photooxidation and generates water-soluble oxygen-containing hydrocarbons. Therefore, here, we investigate the effects of solar irradiation on an asphalt binder. Upon irradiation in a photooxidation microcosm, thin films of the asphalt binder produce abundant oil- and water-soluble oxygenated hydrocarbons, which we hypothesize are also leached from roads and highways through photooxidation reactions. Ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) enables extensive compositional characterization of the virgin asphalt binder, irradiated asphalt binder, and the water-soluble photoproducts. The results reveal the production of water-soluble species that resemble the molecular composition of petroleum-derived dissolved organic matter, including abundant hydrocarbons and S-containing species with up to 18 oxygen atoms. The results also confirm photo-induced oxidation, fragmentation, and potentially polymerization as active processes involved in the production of water-soluble organic pollutants from asphalt.

Probing the Impact of the Knob-into-Hole Mutations on the Structure and Function of a Therapeutic Antibody.

Bispecific antibodies (BsAbs) have drawn increasing interest in the biopharmaceutical industry due to their advantage to bind two distinct antigens simultaneously. The knob-into-hole approach is an effective way to produce bispecific antibodies by driving heterodimerization with mutations in the CH3 domain of each half antibody. To better understand the conformational impact by the knob and hole mutations, we combined size-exclusion chromatography (SEC), differential scanning calorimetry (DSC), and hydrogen-deuterium exchange mass spectrometry (H/D exchange MS), to characterize the global and peptide-level conformational changes. We found no significant alteration in structure or conformational dynamics induced by the knob-into-hole framework, and the conformational stability is similar to the wild-type (WT) IgG4 molecules (except for some small difference in the CH3 domain) expressed in E. coli. Functional studies including antigen-binding and neonatal fragment crystallizable (Fc) receptor (FcRn) binding demonstrated no difference between the knob-into-hole and WT IgG4 molecules in E. coli.

Molecular-Level Characterization of Oil-Soluble Ketone/Aldehyde Photo-Oxidation Products by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Reveals Similarity Between Microcosm and Field Samples.

We present a solid-phase extraction method followed by derivatization with a charged tag to characterize ketone/aldehyde-containing functionalities (proposed photo-oxidation transformation products) in weathered petroleum by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). A photo-oxidation-only microcosm mimics solar irradiation of crude oil in the environment after an oil spill. A biodegradation-only microcosm enables independent determination as to which of the two weathering processes contributes to the formation of oil-soluble ketone/aldehyde species. Results confirm that photo-oxidation produces ketones/aldehydes in crude oil when exposed to solar radiation in laboratory experiments, whereas biodegraded oil samples do not produce ketone/aldehyde compounds. Field samples collected after different time periods and locations after the Deepwater Horizon oil spill are also shown to contain ketones/aldehydes, and comparison of field and photo-oxidation-only microcosm transformation products reveal remarkable similarity. These results indicate that the photo-oxidation microcosm comprehensively represents ketone/aldehyde-formation products in the field, whereas the biodegradation microcosm does not. Solid-phase extraction coupled with derivatization leads to selective identification of ketone/aldehyde species by MS. Although improved dynamic range and slightly reduced mass spectral complexity is achieved by separation/derivatization, comprehensive molecular characterization still requires mass resolving power and mass accuracy provided by FT-ICR MS.

Combating selective ionization in the high resolution mass spectral characterization of complex mixtures.

Direct "dilute and shoot" mass spectral analysis of complex naturally-occurring mixtures has become the "standard" analysis in environmental and petrochemical science, as well as in many other areas of research. Despite recent advances in ionization methods, that approach still suffers several limitations for the comprehensive characterization of compositionally complex matrices. Foremost, the selective ionization of highly acidic (negative electrospray ionization ((-) ESI)) and/or basic (positive electrospray ionization ((+) ESI)) species limits the detection of weakly acidic/basic species, and similar issues (matrix effects) complicate atmospheric pressure photo-ionization (APPI)/atmospheric pressure chemical ionization (APCI) analyses. Furthermore, given the wide range of chemical functionalities and structural motifs in these compositionally complex mixtures, aggregation can similarly limit the observed species to a small (10-20%) mass fraction of the whole sample. Finally, irrespective of the ionization method, the mass analyzer must be capable of resolving tens-of-thousands of mass spectral peaks and provide the mass accuracy (typically 50-300 ppb mass measurement error) required for elemental composition assignment, and thus is generally limited to high-field Fourier transform ion cyclotron mass spectrometry (FT-ICR MS). Here, we describe three approaches to combat the above issues for (+) ESI, (-) ESI, and (+) APPI FT-ICR MS analysis of petroleum samples. Each approach relies on chromatographic fractionation to help reduce selective ionization discrimination and target either specific chemical functionalities (pyridinic and pyrrolic species (nitrogen) or carboxylic acids (oxygen)) or specific structural motifs (single aromatic core (island) or multi-core aromatics (archipelago)) known to be related to ionization efficiency. Each fractionation method yields a 2-10-fold increase in the compositional coverage, exposes species that are undetectable using direct "dilute and shoot" analysis, and provides coarse selectivity in chemical functionalities that can both increase the assignment confidence and optimize ionization conditions to maximize compositional coverage.

Diagnosis of Hemoglobinopathy and ß-Thalassemia by 21 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Tandem Mass Spectrometry of Hemoglobin from Blood.

BACKGROUND: Hemoglobinopathies and thalassemias are the most common genetically determined disorders. Current screening methods include cation-exchange HPLC and electrophoresis, the results of which can be ambiguous because of limited resolving power. Subsequently, laborious genetic testing is required for confirmation. METHODS: We performed a top-down tandem mass spectrometry (MS/MS) approach with a fast data acquisition (3 min), ultrahigh mass accuracy, and extensive residue cleavage by use of positive electrospray ionization 21 Tesla Fourier transform ion cyclotron resonance-tandem mass spectrometry (21 T FT-ICR MS/MS) for hemoglobin (Hb) variant de novo sequencing and ß-thalassemia diagnosis. RESULTS: We correctly identified all Hb variants in blind analysis of 18 samples, including the first characterization of homozygous Hb Himeji variant. In addition, an Hb heterozygous variant with isotopologue mass spacing as small as 0.0194 Da (Hb AD) was resolved in both precursor ion mass spectrum (MS1) and product ion mass spectrum (MS2). In blind analysis, we also observed that the abundance ratio between intact δ and ß subunits (δ/ß) or the abundance ratio between intact δ and α subunits (δ/α) could serve to diagnose ß-thalassemia trait caused by a mutation in 1 HBB gene. CONCLUSIONS: We found that 21 T FT-ICR MS/MS provides a benchmark for top-down MS/MS analysis of blood Hb. The present method has the potential to be translated to lower resolving power mass spectrometers (lower field FT-ICR mass spectrometry and Orbitrap) for Hb variant analysis (by MS1 and MS2) and ß-thalassemia diagnosis (MS1).

Nanostructure of Gasification Charcoal (Biochar).

In this work, we investigate the molecular composition and nanostructure of gasification charcoal (biochar) by comparing it with heat-treated fullerene arc-soot. Using ultrahigh resolution Fourier transform ion-cyclotron resonance and laser desorption ionization time-of-flight mass spectrometry, Raman spectroscopy, and high resolution transmission electron microscopy we analyzed charcoal of low tar content obtained from gasification. Mass spectrometry revealed no magic number fullerenes such as C60 or C70 in the charcoal. The positive molecular ion m/ z 701, previously considered a graphitic part of the nanostructure, was found to be a breakdown product of pyrolysis and not part of the nanostructure. A higher mass distribution of ions similar to that found in thermally treated fullerene soot indicates that they share a nanostructure. Recent insights into the formation of all carbon fullerenes reveal that conditions in charcoal formation are not optimal for the formation of fullerenes, but instead, curved carbon structures coalesce into fulleroid-like structures. Microscopy and spectroscopy support such a stacked, fulleroid-like nanostructure, which was explored using reactive molecular dynamics simulations.

Classification of Plasma Cell Disorders by 21 Tesla Fourier Transform Ion Cyclotron Resonance Top-Down and Middle-Down MS/MS Analysis of Monoclonal Immunoglobulin Light Chains in Human Serum.

The current five-year survival rate for systemic AL amyloidosis or multiple myeloma is ∼51%, indicating the urgent need for better diagnosis methods and treatment plans. Here, we describe highly specific and sensitive top-down and middle-down MS/MS methods owning the advantages of fast sample preparation, ultrahigh mass accuracy, and extensive residue cleavages with 21 telsa FT-ICR MS/MS. Unlike genomic testing, which requires bone marrow aspiration and may fail to identify all monoclonal immunoglobulins produced by the body, the present method requires only a blood draw. In addition, circulating monoclonal immunoglobulins spanning the entire population are analyzed and reflect the selection of germline sequence by B cells. The monoclonal immunoglobulin light chain FR2-CDR2-FR3 was sequenced by database-aided de novo MS/MS and 100% matched the gene sequencing result, except for two amino acids with isomeric counterparts, enabling accurate germline sequence classification. The monoclonal immunoglobulin heavy chains were also classified into specific germline sequences based on the present method. This work represents the first application of top/middle-down MS/MS sequencing of endogenous human monoclonal immunoglobulins with polyclonal immunoglobulins background.

Spontaneous Calcium-Independent Dimerization of the Isolated First Domain of Neural Cadherin.

Cadherins are calcium-dependent, transmembrane adhesion molecules that assemble through direct noncovalent association of their N-terminal extracellular modular domains. As the transmembrane component of adherens junctions, they indirectly link adherent cells' actin cytoskeletons. Here, we investigate the most distal extracellular domain of neural cadherin (N-cadherin), a protein required at excitatory synapses, the site of long-term potentiation. This domain is the site of the adhesive interface, and it forms a dimer spontaneously without binding calcium, a surprising finding given that calcium binding is required for proper physiological function. A critical tryptophan at position 2, W2, provides a spectroscopic probe for the "closed" monomer and strand-swapped dimer. Spectroscopic studies show that W2 remains docked in the two forms but has a different apparent interaction with the hydrophobic pocket. Size-exclusion chromatography was used to measure the levels of the monomer and dimer over time to study the kinetics and equilibria of the unexpected spontaneous dimer formation ( Kd = 130 µM; τ = 2 days at 4 °C). Our results support the idea that NCAD1 is missing critical contacts that facilitate the rapid exchange of the ßA-strand. Furthermore, the monomer and dimer have equivalent and exceptionally high intrinsic stability for a 99-residue Ig-like domain with no internal disulfides ( Tm = 77 °C; Δ H = 85 kcal/mol). Ultimately, a complete analysis of synapse dynamics requires characterization of the kinetics and equilibria of N-cadherin. The studies reported here take a reductionist approach to understanding the essential biophysics of an atypical Ig-like domain that is the site of the adhesive interface of N-cadherin.

Control of Hexamerization, Assembly, and Excluded Strand Specificity for the Sulfolobus solfataricus MCM Helicase.

A growing body of evidence supports a steric exclusion and wrapping model for DNA unwinding in which hexameric helicases interact with the excluded single-stranded DNA (ssDNA) in addition to the encircled strand. Interactions with the excluded ssDNA have been shown to be mediated primarily by electrostatic interactions, but base stacking with surface-exposed tyrosine residues is an alternative hypothesis. Here, we mutated several external tyrosine and positively charged residues from full-length Sulfolobus solfataricus MCM along the proposed path of excluded strand binding and assessed their impact on DNA unwinding. Four of the five tyrosine residues had significant decreases in their level of unwinding, and one, Y519A, located within the α/ß-α linker region of the C-terminal domain, had the most severe perturbation attributed to the disruption of hexamerization. The Y519 mutant exhibits an enhanced and stabilized secondary structure that is modulated by temperature, binding DNA with a higher apparent affinity and suggesting a pathway for hexameric assembly. Hydrogen/deuterium exchange coupled to mass spectrometry was used to map deuterium uptake differences between wild-type and Y519A apo structures highlighting global differences in solvent accessible areas consistent with altered quaternary structure. Two of the five electrostatic mutants had significantly reduced levels of DNA unwinding and combined with previous mutations better define the exterior binding path. The importance of the electrostatic excluded strand interaction was confirmed by use of morpholino DNA substrates that showed analogous reduced unwinding rates. These results better define the hexameric assembly and influence of the excluded strand interactions in controlling DNA unwinding by the archaeal MCM complex.

Middle-Down Characterization of the Cell Cycle Dependence of Histone H4 Posttranslational Modifications and Proteoforms.

Post-translational modifications (PTMs) of histones are important epigenetic regulatory mechanisms that are often dysregulated in cancer. We employ middle-down proteomics to investigate the PTMs and proteoforms of histone H4 during cell cycle progression. We use pH gradient weak cation exchange-hydrophilic interaction liquid chromatography (WCX-HILIC) for on-line liquid chromatography-mass spectrometry analysis to separate and analyze the proteoforms of histone H4. This procedure provides enhanced separation of proteoforms, including positional isomers, and simplifies downstream data analysis. We use ultrahigh mass accuracy and resolution Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometer to unambiguously distinguish between acetylation and tri-methylation (∆m = 0.036 Da). In total, we identify and quantify 233 proteoforms of histone H4 in two breast cancer cell lines. We observe significant increases in S1 phosphorylation during mitosis, implicating an important role in mitotic chromatin condensation. A decrease of K20 unmodified proteoforms is observed as the cell cycle progresses, corresponding to an increase of K20 mono- and di-methylation. Acetylation at K5, K8, K12, and K16 declines as cells traverse from S phase to mitosis, suggesting cell cycle-dependence and an important role during chromatin replication and condensation. These new insights into the epigenetics of the cell cycle may provide new diagnostic and prognostic biomarkers.

Mechanistic Origins of Enzyme Activation in Human Glucokinase Variants Associated with Congenital Hyperinsulinism.

Human glucokinase (GCK) acts as the body's primary glucose sensor and plays a critical role in glucose homeostatic maintenance. Gain-of-function mutations in gck produce hyperactive enzyme variants that cause congenital hyperinsulinism. Prior biochemical and biophysical studies suggest that activated disease variants can be segregated into two mechanistically distinct classes, termed α-type and ß-type. Steady-state viscosity variation studies indicate that the kcat values of wild-type GCK and an α-type variant are partially diffusion-limited, whereas the kcat value of a ß-type variant is viscosity-independent. Transient-state chemical quench-flow analyses demonstrate that wild-type GCK and the α-type variant display burst kinetics, whereas the ß-type variant lacks a burst phase. Comparative hydrogen-deuterium exchange mass spectrometry of unliganded enzymes demonstrates that a disordered active site loop, which folds upon binding of glucose, is protected from exchange in the α-type variant. The α-type variant also displays an increased level of exchange within a ß-strand located near the enzyme's hinge region, which becomes more solvent-exposed upon glucose binding. In contrast, ß-type activation causes no substantial difference in global or local exchange relative to that of unliganded, wild-type GCK. Together, these results demonstrate that α-type activation results from a shift in the conformational ensemble of unliganded GCK toward a state resembling the glucose-bound conformation, whereas ß-type activation is attributable to an accelerated rate of product release. This work elucidates the molecular basis of naturally occurring, activated GCK disease variants and provides insight into the structural and dynamic origins of GCK's unique kinetic cooperativity.

Linking Natural Oil Seeps from the Gulf of Mexico to Their Origin by Use of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

We report chemical characterization of natural oil seeps from the Gulf of Mexico by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and Gas Chromatography/Atmospheric Pressure Chemical Ionization Mass Spectrometry (GC/APCI-MS), to highlight how FT-ICR MS can also be employed as a means to determine petroleum connectivity, in addition to traditional GC/MS techniques. The source of petroleum is the Green Canyon (GC) 600 lease block in the Gulf of Mexico. Within GC600, two natural oil seepage zones, Mega Plume and Birthday Candles, continuously release hydrocarbons and develop persistent oil slicks at the sea surface above them. We chemically trace the petroleum from the surface oil slicks to the Mega Plume seep itself, and further to a petroleum reservoir 5 km away in lease block GC645 (Holstein Reservoir). We establish the connectivity between oil samples and confirm a common geological origin for the oil slicks, oil seep, and reservoir oil. The ratios of seven common petroleum biomarkers detected by GC/APCI-MS display clear similarity between the GC600 and GC645 samples, as well as a distinct difference from another reservoir oil collected ∼300 km away (Macondo crude oil from MC252 lease block). FT-ICR MS and principal component analysis (PCA) demonstrate further similarities between the GC600 and GC645 samples that distinctly differ from MC252. A common geographical origin is postulated for the GC600/GC645 samples, with petroleum migrating from the GC645 reservoir to the oil seeps found in GC600 and up through the water column to the sea surface as an oil slick.