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Application of FT-ICR Mass Spectrometry in Study of Proteomics, Petroleomics and Fragmentomics

Title: Application of FT-ICR Mass Spectrometry in Study of Proteomics, Petroleomics and Fragmentomics.
Name(s): Mao, Yuan, author
Marshall, Alan G., professor directing dissertation
Blaber, Michael, university representative
Dalal, Naresh S., committee member
Roper, Michael G., committee member
Department of Chemistry and Biochemistry, degree granting department
Florida State University, degree granting institution
Type of Resource: text
Genre: Text
Issuance: monographic
Date Issued: 2013
Publisher: Florida State University
Florida State University
Place of Publication: Tallahassee, Florida
Physical Form: computer
online resource
Extent: 1 online resource
Language(s): English
Abstract/Description: With the advent of two "soft" ionization techniques in the late 1980s—electrospray ionization (ESI) and matrix assisted laser desorption/ionization (MALDI) for the routine and general formation of intact molecular ions—and continuing effort in the instrumentation development directed toward improving two key parameters of mass spectrometric performance—mass resolving power and mass accuracy, mass spectrometry has been an indispensable analytical technique for chemical and biochemical sample analysis, especially for highly complex mixture systems, e.g., proteomics, petroleomics, lipidomics, metabolomics, etc. The introduction of ESI and MALDI ionization techniques has extended the accessibility of mass spectrometry-based analysis from small volatile molecules to large non-volatile molecules, whereas mass accuracy and mass resolving power directly determine the usefulness of mass spectrometric experiments. Among the high resolution mass analyzers such as reflectron/multipass time of flight (TOF), orbitrap, and Fourier transform ion cyclotron resonance (FT-ICR), FT-ICR MS provides ten-fold higher mass resolution and mass measurement accuracy than other mass analyzers and has become the most powerful techniques that can deal with the complexity of various samples, e.g., it is possible to routinely achieve high mass resolving power of 400,000 (m/∆m50% ≈ 400,000, in which m is molecular mass and ∆m50% is the mass spectral peak width at half-maximum peak height) and mass accuracy (~ 100 ppb) up to 800 Da from high-field (≥9.4 T) FT-ICR MS, thus resolving > 40,000 different elemental compositions in a single mass spectrum and providing their unambiguous molecular formulas. Chapter 1 introduces the concept of mass resolving power, mass resolution and mass measurement accuracy, the principle of the FT-ICR instrument for mass measurement, ionization methods, factors that control the mass measurement accuracy of FT-ICR MS and utility of high mass accuracy for analysis of biological samples and complex mixtures. Chapter 2 describes the application of high mass accuracy for distinction of N-terminal and C-terminal electron capture dissociation/electron transfer dissociation (ECD/ETD) product ions of c and z⋅ based on their number of hydrogen plus nitrogen atoms determined by accurate mass measurement, and forms a basis for de novo peptide sequencing. The effect of mass accuracy (0.1-1 ppm error) on c/z⋅ overlap and unique elemental composition overlap is evaluated for a database of c/z⋅ product ions each based on all possible amino acid combinations and four subset databases containing the same c ions but with z⋅ ions determined by in silico digestion with trypsin, Glu-C, Lys-C, or chymotrypsin. High mass accuracy reduces both c/z⋅ overlap and unique elemental composition overlap. Of the four proteases, trypsin offers slightly better discrimination between N- and C-terminal ECD/ETD peptides. Interestingly, unique elemental composition overlap curves for c/c and z⋅/z⋅ peptide ions exhibit discontinuities at certain nominal masses for 0.1-1.0 ppm mass error. Also, the number of ECD/ETD product ion amino acid compositions as a function of nominal mass increases exponentially with mass, but with a superimposed modulation due to higher prevalence of certain elemental compositions. Chapter 3 presents an idea that at sufficiently high mass accuracy, it is possible to distinguish phosphorylated from unmodified peptides by mass measurement alone. We examine the feasibility of that idea, tested against a library of all possible in silico tryptic digest peptides from the human proteome database. The overlaps between in silico tryptic digest phosphopeptides generated from known phosphorylated proteins (1-12 sites) and all possible unmodified human peptides are considered for assumed mass error ranges of ±10, ±50, ±100, ±1,000, and ±10,000 ppb. We find that for mass error ±50 ppb, 95% of all phosphorylated human tryptic peptides can be distinguished from nonmodified peptides by accurate mass alone through the entire nominal mass range. We discuss the prospect of on-line LC MS/MS to identify phosphopeptide precursor ions in MS1 for selected dissociation in MS2 to identify the peptide and site(s) of phosphorylation. ETD and ECD are two similar fragmentation approaches, producing extensive and nonspecific fragmentation (c/z⋅ ions formed by cleavage of N-Cα backbone bond) while retaining thermally labile post-translational modifications. In chapter 4, we implemented dual electrospray ionization ETD on a custom-built 9.4 T FT-ICR MS. Two separate electrospray emitters are automatically switched for injection of positive (analyte) and negative (reagent) ions. Decarboxylated 2-(fluoranthene-8-carbonyl) benzoic acid is the ETD reagent anion. A linear octopole ion trap is the ETD ion/ion reaction chamber, and an RF voltage is applied to the front and back ion trap electrodes to confine both cations and reagent anions for ETD, after which the c- and z- type product ions are passed to the ICR cell for high resolution and mass accuracy analysis. Comparison of ETD and ECD spectra of standard peptides shows that ETD provides similar sequence coverage and fragmentation pattern to ECD. Chapter 5 describes a calibration procedure in which accurate masses of spacings from any two same type neighboring fragment ions differing by one amino acid residue are used to calibrate ECD and collision activated dissociation (CAD) MS/MS spectra of standard peptides with different molecular weights and charge states. High mass accuracy of tandem mass spectra is crucial for confident extraction and identification of spacings. FT-ICR mass spectrometry provides ultrahigh mass accuracy and resolving power and acquired MS/MS spectra with ppm mass accuracy level are routinely obtained when combined with external calibration by substance P fragments. Calibration by accurate masses of extracted spacings shows up to ~ 30% further reduction of rms mass error of MS/MS spectra on average compared with substance P MS/MS external calibration. ~ 25% improvement of c/z⋅ ion unambiguous distinction from ECD spectra based on valence parity rule increases the confidence of peptide sequencing. FT-ICR MS with ultrahigh resolving power and mass accuracy is essential to resolve and uniquely identify elemental compositions of thousands of components in complex organic mixtures, e.g., petroleum crude oils. To study how much resolving power and mass accuracy is necessary, in chapter 6 all possible closest mass doublets (0<∆m<45 mDa, ∆m is the mass difference of mass doublet) were counted for both electrospray ionization (ESI) and atmospheric pressure photoionization (APPI) absorption-mode spectra automatically by use of the algorithm written in LabWindows/CVI. As many as thousands of mass doublets with the mass difference less than 10 mDa (as low as ~ 0.70 mDa) were observed in APPI and ESI absorption-mode broadband mass spectra. Histograms of mass doublet distribution for APPI and ESI were plotted. In chapter 7 the effect of mass error (10 - 500 ppb) on elemental composition overlap is evaluated for ESI and APPI databases containing all possible elemental compositions with proper constraints, CcHhNnOoSs13Ccc34Sss, c, h unlimited, 0≤n<5, 0≤o<10, 0≤s≤3, 0≤cc<3 and 0≤ss<2 for even-electron ions (M+H)+ (or (M-H)-) in ESI and for both M+⋅ and (M+H)+ (or M-⋅ and (M-H)-) in APPI with nominal mass of 200-1200 Da. Number of element compositions of all possible components in complex mixtures is reduced by ~ 1100 on average for each class after applying 90% rule. High mass accuracy reduces elemental composition overlap and facilitates the unique identification of elemental compositions for components up to 1200 Da at mass errors of 200 ppb in ESI and 100 ppb in APPI. All possible theoretical mass doublets which may occur in petroleum crude oils are calculated based on elemental compositions of all possible components from ESI and APPI databases. Mass doublets with mass difference as low as 0.20 mDa, even smaller than mass of electron (0.548 mDa) and NO213C vs. C2H3S (0.71 mDa) (the smallest one currently observed in 9.4 T broadband absorption-mode ESI/APPI FT-ICR MS) are calculated and can only be resolved with higher-field FT-ICR MS (e.g., absorption-mode 21T). FTICR mass spectrometer coupled with ECD/ETD offers ultrahigh broadband mass resolving power (>105) and mass accuracy (<1 ppm) for detection of accurate precursor mass as well as the vast amount of isotopically resolved fragment ions required for protein identification and has become an increasing useful tool for top-down analysis. In chapter 8 we perform top-down ECD FT-ICR MS for structural analysis of an intact monoclonal antibody (IgG1-kappa (κ) isotype, ~148 kDa). Simultaneous ECD for all charge states (42+ to 58+) generates more extensive cleavages than ECD for an isolated single charge state. The cleavages are mainly localized in the variable domains of both heavy and light chains, the respective regions between the variable and constant domains in both chains, the region between heavy chain constant domains CH2 and CH3, and the disulfide bond (S-S) linked heavy chain constant domain CH3. The light chain yields mainly N-terminal fragment ions due to the protection of the inter-chain disulfide bond between light and heavy chain, and limited cleavage sites are observed in the variable domains for each chain where the S-S spans the polypeptide backbone. Only a few cleavages in the S-S linked light chain constant domain, hinge region, and heavy chain constant domains CH1 and CH2 are observed, leaving glycosylation uncharacterized. Top-down ECD with a custom-built 9.4 T FT-ICR MS for structural characterization of IgG1κ provides more extensive sequence coverage than top-down collision induced dissociation (CID) and ETD with time-of-flight but comparable sequence coverage with top-down ETD with orbitrap MS.
Identifier: FSU_migr_etd-7489 (IID)
Submitted Note: A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Degree Awarded: Spring Semester, 2013.
Date of Defense: April 1, 2013.
Keywords: Fragmentomics, FT-ICR MS, Mass accuracy, Mass resolution, Petroleomics, Proteomics
Bibliography Note: Includes bibliographical references.
Advisory Committee: Alan G. Marshall, Professor Directing Dissertation; Michael Blaber, University Representative; Naresh S. Dalal, Committee Member; Michael G. Roper, Committee Member.
Subject(s): Chemistry
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Mao, Y. (2013). Application of FT-ICR Mass Spectrometry in Study of Proteomics, Petroleomics and Fragmentomics. Retrieved from