High-field (9.4 T) Fourier transform ion cyclotron resonance (FT-ICR)
mass spectra of standard
Suwannee River humic and fulvic acids have been obtained by use of
laser desorption (LDI) and
electrospray (ESI) ionization. The LDI FT-ICR mass spectrum was
similar to those observed
previously, with ions at essentially every nominal value, 200 ≤
m/z ≤ 800. In contrast, the
ESI
FT-ICR mass spectrum, although still containing ions at most values in
the 200 ≤ m/z ≤ 800
range, was dominated by a relatively few prominent species. ESI
FT-ICR mass spectra of standard
humic and fulvic acid isolates were similar. Although many ionic
species appeared in both fulvic
acid and humic acid ESI FT-ICR mass spectra, the fulvic acid mass
spectrum contained more
highly charged species. Subfractions of the fulvic acid mixture
isolated by an HPLC procedure
yielded similar mass spectra. The stability of high-mass ions
produced by ESI combined with
the high-mass resolution capability of FT-ICR MS allow for precise
determination of molecular
masses, from which molecular formulas may be obtained by mass alone.
Future two-dimensional
FT-ICR MS2 determinations of humic and fulvic acid
structures should be feasible by use of
collisionally induced and multiple-photon dissociation
techniques.
Significantly improved sensitivity for analysis of biomolecules by MALDI FT-ICR mass spectrometry is achieved by (i) microscope-monitored sample deposition onto a small indentation on the probe tip and (ii) multiple remeasurement of ions from a single laser shot. A simple modification to the solids probe tip allows for microdeposition of a few amols of analyte onto small indentation spots previously aligned with the laser beam. Ion multiple remeasurement of the same ion packet enhances the signal-to-noise ratio and thus extends the achievable FT-ICR MS detection limit. We demonstrate that FT-ICR can be used to detect parent and structurally significant fragment ions of peptides and phospholipids at low amol amounts. Positive ion mass spectra for approximately 90 amol of a mixture of angiotensin II and bradykinin, approximately 40 amol of dipalmitoylglycerophosphatidylcholine, and approximately 8 amol of substance P constitute the lowest reported detection limits to date for FT-ICR mass analysis of MALDI-generated ions.
A gas-phase hydrogen/deuterium (H/D) exchange reaction technique to determine the number of active hydrogens (NOAH) in fulvic acid ions using electrospray
ionization Fourier transform ion cyclotron resonance mass
spectrometry is described. First, fulvic acid precursor ions
are isolated by stored waveform inverse Fourier transform
dipolar excitation. Second, ion−molecule reactions of the
selected fulvic acid ions with neutral H/D exchange
reagent gases are monitored. The number of incorporated
deuterium isotopes in the product ion provides the NOAH
for the precursor ion. Previously characterized northern
hardwood (NHFA) and red spruce (NCFA) fulvic acid
samples were analyzed in this study. Selected ions of both
fulvic acid samples undergo H/D exchange with D2O,
ND3, and CD3OD reagent gases. The extent of H/D
exchange of the fulvic acid ions increases with reagent gas
basicity, D2O < CD3OD < ND3. For the first time, we were
able to count the NOAH of selected fulvic acid molecules.
The average maximum NOAH for NCFA and NHFA ions
at m/z region 700−1000 Th is ∼7−9. For example, the
singly charged NCFA positive ions at m/z 800 Th contain
eight active hydrogens. There is no significant difference
between NOAH for NCFA and NHFA. The proton affinities
of fulvic acid ions at m/z range of 600−1000 Th do not
vary significantly.
By combined and repeated use of sustained off-resonance irradiation (SORI) for ion dissociation, stored waveform inverse Fourier transform (SWIFT) waveforms for ion isolation, and ion axialization and remeasurements techniques, we obtain for the first time MS, MS2, and MS3 FT-ICR mass spectra from peptide ions (enzymatic digest products of horse cytochrome c) produced from a single laser shot. The successive fragmentation of gas-phase ions detected from the same initial batch of ions increases the sensitivity of analysis of trace amounts of biological samples in structural mass spectrometry, and fragment identification is facilitated by resolution of carbon-13 isotopic distributions. The method is illustrated by analyses of subfemtomole amounts of crudely purified samples of tryptic digest solutions of horse cytochrome c and bovine cytochrome c. The high-resolution primary ion mass spectrum, along with the collision-induced dissociation (CID) and MSn capabilities of FT-ICR, help to determine the primary amino acid sequence of the fragment ions beyond what is obtained from enzymatic digestion alone, without prior chromatographic separation and purification.
We utilized gas phase hydrogen/deuterium (H/D) exchange reactions and ab initio calculations to investigate the complexation between a model peptide (Arg-Gly-Asp[triple bond]RGD) with various alkali metal ions. The peptide conformation is drastically altered upon alkali metal ion complexation. The associated conformational changes depend on both the number and type of complexing alkali metal ions. Sodium has a smaller ionic diameter and prefers a multidentate interaction that involves all three amino acids of the peptide. Conversely, potassium and cesium form different types of complexes with the RGD. The [RGD + 2Cs - H]+ species exhibit the slowest H/D exchange reactivity (reaction rate constant of approximately 6 x 10(-13) cm3molecule(-1)s(-1) for the fastest exchanging labile hydrogen with ND3). The reaction rate constant of the protonated RGD is two orders of magnitude faster than that of the [RGD + 2Cs - H]+. Addition of the first cesium to the RGD reduces the H/D exchange reaction rate constant (i.e., D0) by a factor of seven whereas sodium reduces this value by a factor of thirty. Conversely, addition of the second alkali metal ions has the opposite effect; the rate of D0 disappearance for all [RGD + 2Met - H]+ species (Met[triple bond]Na, K, and Cs) decreases with the alkali metal ion size.
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