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2005 ASMS Posters
ThP517
ThP580
TP551
ThP279
Journal Articles
Matrix Influence on the Formation of Positively Charged Oligonucleotides in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry
Chau-Wen Chou, Peter Williams1 and Patrick A. Limbach
Department of Chemistry, Louisiana State University, Baton Rouge LA
1 Department of Chemistry, Arizona State University, Tempe AZ
Abstract
The ionization efficiency of various ultraviolet matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) matrices was investigated. A site of fixed positive charge was generated on an oligonucleotide by addition of a quaternary ammonium. This quaternary ammonium-tagged oligonucleotide was then used as an internal standard to probe the relative ionization capabilities of 3-hydroxypicolinic acid (3-HPA), 2',4',6'-trihydroxyacetophenone (THAP) and 2,5-dihydroxybenzoic acid (DHBA) in positive-ion mode. MALDI-MS analysis of equimolar mixtures of the quaternary ammonium-tagged oligonucleotide and an unmodified polythymidylic acid, dT12, found that 3-HPA yielded more abundant protonated dT12 molecular ions that either THAP or DHBA. These results demonstrate that the low ion yields previously reported for polythymidylic acid are due to the matrix utilized and are not due to the low proton affinity value of thymidine. Primary, secondary and tertiary amines were also incorporated into dT12 to examine the effect of these different amines on the protonation efficiency of the three matrices under investigation. Similar results were obtained, regardless of the amine-tag utilized, with protonation efficiency following the trend 3-HPA > THAP > DHBA. Consideration of the various factors which might influence the overall production of positively charged polythymidylic acid finds that it is the matrix:phosphodiester backbone interaction that might play the important role in determining optimal MALDI-MS response. These results are a step towards understanding the matrix properties necessary for optimal production of oligonucleotide molecular ions in MALDI-MS.
Influence of Ionization Energy on Charge-Transfer Ionization in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry
Stephen F. Macha, Tracy D. McCarley and Patrick A. Limbach
Department of Chemistry, Louisiana State University, Baton Rouge LA
Abstract
In this study, non-polar matrixes are used in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to analyze selected non-polar analytes. Our assumption is that gas-phase charge-transfer reactions between matrix and analyte are responsible for generation of analyte radical molecular ions. Following this assumption, the ionization energies of the matrixes and analytes should have a direct influence on the production of radical molecular cations of the analytes. To test this assumption, several non-polar analytes including ferrocene and ferrocene derivatives, trans-stilbene, triphenylphosphine, 2,2'-methylenebis(6-tert-butyl-4-methylphenol), biphenyl and 1,4-bis(methylthio)benzene were studied using positive-ion mode MALDI-TOFMS. The results of these studies demonstrate that formation of the radical molecular cation is dependent on the difference in ionization energies between the matrix and the analyte. The propensity for charge-transfer ionization, as opposed to proton-transfer ionization, for these analytes was confirmed using atmospheric pressure chemical ionization mass spectrometry. Charge-transfer ionization using non-polar matrixes in MALDI-MS is a suitable method for the characterization of a number of non-polar, thermally labile analytes.
Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry of Hydrophobic Peptides
Kari B. Green-Church and Patrick A. Limbach
Department of Chemistry, Louisiana State University, Baton Rouge LA
Abstract
Hydrophobic peptides, especially those with acid-labile protecting groups, are difficult to characterize using mass spectrometric methods. We have developed a new procedure for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of such samples. Hydrophobic peptides, which are insoluble in aqueous solutions, are dissolved in chloroform and combined with matrixes prepared in chloroform or chloroform/methanol solutions. The use of a common solvent for the matrix and the analyte improves the analyte isolation step in MALDI mass spectrometry. The lack of acidic solutions previously used for electrospray ionization- or MALDI-mass spectrometry of hydrophobic peptides extends this methodology to cyclic or protected hydrophobic peptides. Conventional peptide matrixes, such as 2,5-dihydroxybenzoic acid and sinapinic acid, as well as 3-indoleacrylic acid are shown to be suitable for hydrophobic peptides. Cyclic hydrophobic peptides and linear hydrophobic peptides with blocked termini are detected as the cation-adducted pseudomolecular ion due to the lack of suitable sites of protonation on the analyte.
Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry: An Overview
Patrick A. Limbach
Department of Chemistry, Louisiana State University, Baton Rouge LA
Abstract
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry burst on the scene nearly ten years ago and has since revolutionized the field of mass spectrometry. MALDI-MS is a viable mass spectrometric approach for the characterization of biomolecules, synthetic polymers, petroleum and coal products, inorganic and organic materials. The simplicity, speed and performance features of MALDI-MS permit the characterization of compounds previously intractable to mass spectrometric analysis.
Electron Transfer Ionization in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry
Tracy D. McCarley, Robin L. McCarley and Patrick A. Limbach
Department of Chemistry, Louisiana State University, Baton Rouge LA
Abstract
In this paper we present time-of-flight mass spectral data demonstrating matrix-assisted laser desorption/electron-transfer ionization of several analytes. The matrices used here to achieve electron-transfer ionization are terthiophene and anthracene, both of which in positive mode form only molecular radical cations (M+.) upon laser irradiation (l=337 nm) at near-threshold laser powers. Analytes studied include metallocenes (1,2-diferrocenylethane, ferrocene, and decamethylferrocene) and a methylene-bridged bisphenol, 2,2'-methylenebis(6-tert-butyl-4-methylphenol). In the mass spectra of these matrix/analyte combinations, the formation of protonated molecules was not observed. Instead, each analyte formed a molecular radical cation (A+.) when either matrix was used. Experiments utilizing anthracene-d10 as the matrix confirmed the formation of only the analyte molecular radical cation. In addition, the molecular radical cation of ferrocene - not the protonated molecule - was produced when 2,5-dihydroxybenzoic acid was used as the matrix, indicating that a matrix commonly used to form protonated polar analytes can, in addition, function as an electron-transfer MALDI matrix.
The Influence of Co-matrix Proton Affinity on Oligonucleotide Ion Stability in Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
Tracey A. Simmons and Patrick A. Limbach
Department of Chemistry, Louisiana State University, Baton Rouge LA
Abstract
In this paper we investigated the role organic base co-matrices play in reducing oligonucleotide fragmentation during analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The organic base co-matrix plays an important role in influencing the gas-phase behavior of desorbed oligonucleotides. No correlation was found between the solution pH values and the molecular ion stability of two model oligonucleotides. Instead, a direct correlation between the co-matrix proton affinity and the oligonucleotide molecular ion stability is seen. A co-matrix whose proton affinity is close to or greater than the proton affinity of the nucleobases can serve as a "proton sink". We propose that upon laser desorption/ionization, the co-matrix competes with the nucleobases of the oligonucleotide for additional protons from the matrix. When a co-matrix, such as triethylamine, is added, the co-matrix, rather than the oligonucleotide nucleobases, is the preferred site of proton transfer from the matrix. Titration of standard oligonucleotide matrices with several co-matrices of differing proton affinity demonstrates that the co-matrix mole fraction is an important factor in oligonucleotide molecular ion stability. When the mole fraction of the co-matrix approaches that of the matrix, nearly complete elimination of oligonucleotide fragmentation is seen. Control experiments utilizing pyridine, a co-matrix whose proton affinity is less than that of thymine or the phosphodiester backbone, demonstrate that the co-matrix plays an active role in oligonucleotide stabilization.
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The Use of a Co-matrix for Improved Analysis of Oligonucleotides by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
Tracey A. Simmons and Patrick A. Limbach
Department of Chemistry, Louisiana State University, Baton Rouge LA
Abstract
Cation adduction to oligonucleotides and nucleic acids during mass spectrometric analysis is a recurrent problem. We have that the use of organic base solutions of high gas-phase proton affinity, as used previously in electrospray ionization mass spectrometry, can significantly reduce the cation adduction problem during matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. A comparison of the utility of adding imidazole or triethylamine as a co-matrix versus the standard addition of cation-exchange resin beads was made. The co-matrices studied were found to be more effective than the cation exchange resin beads at reducing cation adducts from samples containing a high level of salt. The use of co-matrices also appears to improve the gas-phase stability of larger oligonucleotides. MALDI-time-of-flight analysis of a binary mixture of oligoribonucleotide 27-mers with a co-matrix exhibited less metastable decomposition than the same analysis using cation exchange resin beads. The use of co-matrices may be a viable strategy for the mass spectrometric characterization of larger oligonucleotides obtained from media containing a high salt content such as polymerase chain reaction products.
Chemical Sequencing of Phosphorothioate Oligonucleotides Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
Lenore M. Polo, Tracy D. McCarley and Patrick A. Limbach
Department of Chemistry, Louisiana State University, Baton Rouge LA
Abstract
A new technique for sequencing phosphorothioate oligonucleotides is demonstrated that uses matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Current sequencing protocols for phosphorothioate analysis have drawbacks to their widespread implementation. Enzymatic and Maxam-Gilbert sequencing techniques require oxidation to the phosphodiester moiety, thereby increasing analysis time and eliminating the ability to locate modified linkages. Tandem mass spectrometry, which is finding increased use in phosphodiester oligonucleotide sequencing, has not been demonstrated on molecules with phosphorothioate-linked backbones. In the approach presented here, sequencing of phosphorothioate-linked oligonucleotide is carried out using 2-iodoethanol to cleave at the thiolated sites along the backbone. The technique has several advantages over current phosphorothioate sequencing methods: (1) sequencing is performed on the sample without prior oxidation; (2) both 5'-3' and 3'-5' mass ladders are generated, permitting bidirectional sequencing; (3) it is possible to determine the location of the phosphorothioate linkages in mixed phosphorothioate/phosphodiester oligonucleotides; (4) analysis times are short, (<90 s); and (5) small sample amounts are used (<10 nmol). This approach is demonstrated on oligonucleotides with phosphorothioate linkages and should be amenable to the analysis of phosphorodithioates.
Indirect Mass Spectrometric Methods for Characterizing and Sequencing Oligonucleotides
Patrick A. Limbach
Department of Chemistry, Louisiana State University, Baton Rouge LA
Abstract
The use of mass spectrometry for the characterization and sequence determination of oligonucleotides is reviewed. This review focuses primarily on the use of mass spectrometry to analyze sequence-specific fragments of oligonucleotides that are generated via solution-phase chemical reactions. The majority of these "indirect" sequencing methods are a result of recent advances in electrospray ionization and matrix-assisted laser desorption/ionization for the generation of intact gas-phase ions from oligonucleotides. Descriptions of the current indirect sequencing protocols will be presented as well as a comparison of the applicability of these procedures for analyzing "real world" samples. The applicability of indirect mass spectrometric sequencing to antisense oligonucleotides will be discussed in detail.
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