David J. Aaserud

193 nm photodissociation of larger multiply-charged biomolecules

Abstract In contrast to most ion dissociation methods, 193 nm ultraviolet photodissociation of electrosprayed melittin (2.8 kDa) and ubiquitin (8.6 kDa) molecular ions yields new c and z ions (backbone amine bond dissociation) that provide additional sequence information. Dissociation by collisions or infrared photons produce b and y ions; for cleavages between the same amino acids the c ion represents the addition of NH 2 to the b ion, and z the loss of NH 2 from the y ion, so that these ions can be differentiated by this ± 16.02 Da difference. However, 193 nm photodissociation of 12–29 kDa ions as yet does not give collectable product ions, and that of the very stable y 18 2+ ion from ubiquitin only effects a side chain loss. 193 nm irradiation of negative ions of all-T 30-mer DNA appears to eject electrons; apparently this is the first observation of electron photodetachment from multiply-charged negative ions.

Accurate base composition of double-strand DNA by mass spectrometry

An accurate molecular weight (Mr) assignment for a double-strand (ds) DNA determines or greatly restricts the possible number of each of its four bases, while the compositions for its two single-strand (ss) components can also be derived from their Mr values. For a ds 64-mer (39 kDa), the ss-Mr values (±0.5 Da) of its high-resolution mass spectrum from an electrospray ionization/Fourier transform instrument yield only the correct ds- and ss-base compositions. Literature mass spectra of lower mass accuracy show that such data can also restrict their possible composition assignments, with further discrimination using the abundance vs. base composition of small fragment ions from the dissociation of the ss molecular ions.

Gel permeation chromatography coupled to fourier transform mass spectrometry for polymer characterization.

We report an on-line coupling of gel permeation chromatography (GPC) to Fourier transform mass spectrometry (FTMS) using a modified commercial electrospray ionization (ESI) interface. Selected oligomer profiles for the sodiated (1+ through 5+ charge states) oligomer ions of a narrow-molecular-weight poly(methyl methacrylate) were generated and used for obtaining a calibration curve. Using the MS-generated calibration curve and the refractive index response for quantification, an accurate molecular weight distribution was calculated and showed an excellent agreement with the value specified by the supplier. GPC/ESI/FTMS also allowed for an unequivocal end-group determination and characterization of a secondary distribution due to the formation of cyclic reaction products. We analyzed a glycidyl methacrylate/butyl methacrylate copolymer with a broad molecular weight distribution, where fractionation and high resolving power were required for adequate characterization. Molecular weight distribution data showed the advantage of coupling high-resolution MS and GPC to overcome the difficulty of analyzing polydisperse polymers with MS alone.

Identification, Composition, and Asymmetric Formation Mechanism of Glycidyl Methacrylate/Butyl Methacrylate Copolymers up to 7000 Da from Electrospray Ionization Ultrahigh-Resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Glycidyl methacrylate (GMA) and butyl methacrylate (BMA) have the same nominal mass (142 Da) but differ in exact mass by 0.036 Da (CH(4) vs O). Therefore, copolymers formed from the two isobaric monomers exhibit a characteristic isobaric distribution due to different monomer compositions. Here, we show that electrospray ionization FT-ICR mass spectrometry at 9.4 T resolves the isobaric components of copolymers as large as 7000 Da with a resolving power (m/Δm(50%)) of ∼500 000 in a gel permeation chromatography fractionated polymer sample. That resolution provides for complete and unequivocal component analysis of such copolymers of the size used for high solid content automobile coatings. All five possible copolymer products predicted by the polymerization mechanism are resolved and identified in the mass spectrum. Two of those polymer series (each with saturated end group) were previously unresolved by mass spectrometry because they differ in mass from the two other unsaturated products by only 0.0089 Da. Finally, analysis of the asymmetrical isobaric distribution for the copolymer n-mers, (GMA)(m)(BMA)(n)(-)(m), 0≤ m ≤ n, in which species with adjacent values of m differ from each other in mass by 36 mDa (i.e., the mass difference, CH(4) vs O, between GMA and BMA) proves that GMA is less reactive than BMA in the polymerization process.

DNA sequencing with blackbody infrared radiative dissociation of electrosprayed ions

Abstract As shown by Williams for positive multiply-charged ions, negative electrosprayed ions trapped in a Fourier-transform ion cyclotron resonance mass spectrometer can be dissociated efficiently with BIRD. For a 50-mer DNA, an ion cell wall temperature of 90°C gives no dissociation and 120°C gives only uninformative base loss. However, 150°C gives extensive dissociation producing ionic products qualitatively similar to those from laser IR, collisionally activated, and nozzle-skimmer dissociation. Under the conditions used, BIRD produces more fragment ions that have lost an additional base (AH). Structurally informative BIRD spectra are also observed for a 100-mer DNA and a double-strand 64-mer DNA. For both samples, BIRD successfully ‘boiled off’ non-covalent adducts that produced an uninterpretable spectrum, while laser IR was only successful in this for the ds 64-mer.

Double stranded DNA sequencing by tandem mass spectrometry

Abstract Electrospray ionization produces far more abundant molecular ions for double stranded (ds) DNA than for single stranded (ss) and accurate molecular masses can provide the base composition of dsDNA. This study shows that the Fourier-transform mass spectra of a ds 64-mer DNA can provide approximately 50% more sequence information than the spectra of either individual ss component. Cleavages triggered by T loss are of low probability, but the opposite is true in the complementary ss strand that bears the base A at this site. The spectrum of the product of the attempted biological synthesis of a ds 70-mer DNA provided critical information on its extensive 3′-end heterogeneity.