16 research outputs found
Selective Gas-Phase Ion/Ion Reactions: Enabling Disulfide Mapping via Oxidation and Cleavage of Disulfide Bonds in Intermolecularly-Linked Polypeptide Ions
The selective gas-phase oxidation
of disulfide bonds to their thiosulfinate
form using ion/ion reactions and subsequent cleavage is demonstrated
here. Oxidizing reagent anions are observed to attach to all polypeptides,
regardless of amino acid composition. Direct proton transfer yielding
a charge-reduced peptide is also frequently observed. Activation of
the ion/ion complex between an oxidizing reagent anion and a disulfide-containing
peptide cation results in oxygen transfer from the reagent anion to
the peptide cation to form the [M+H+O]<sup>+</sup> species. This thiosulfinate
derivative can undergo one of several rearrangements that result in
cleavage of the disulfide bond. Species containing an intermolecular
disulfide bond undergo separation of the two chains upon activation.
Further activation can be used to generate more sequence information
from each chain. These oxidation ion/ion reactions have been used
to illustrate the identification of S-glutathionylated and S-cysteinylated
peptides, in which low molecular weight thiols are attached to cysteine
residues in peptides via disulfide bonds. The oxidation chemistry
effectively labels peptide ions with readily oxidized groups, such
as disulfide bonds. This enables a screening approach for the identification
of disulfide-linked peptides in a disulfide mapping application involving
enzymatic digestion. The mixtures of ions generated by tryptic and
peptic digestions of lysozyme and insulin, respectively, without prior
separation or isolation were subjected both to oxidation and proton
transfer ion/ion chemistry to illustrate the identification of peptides
in the mixtures with readily oxidized groups
Ion/Ion Reactions of MALDI-Derived Peptide Ions: Increased Sequence Coverage via Covalent and Electrostatic Modification upon Charge Inversion
Atmospheric pressure matrix-assisted laser desorption/ionization
(AP-MALDI)-derived tryptic peptide ions have been subjected to ion/ion
reactions with doubly deprotonated 4-formyl-1,3-benzenedisulfonic
acid (FBDSA) in the gas-phase. The ion/ion reaction produces a negatively
charged electrostatic complex composed of the peptide cation and reagent
dianion, whereupon dehydration of the complex via collision-induced
dissociation (CID) produces a Schiff base product anion. Collisional
activation of modified lysine-terminated tryptic peptide anions is
consistent with a covalent modification of unprotonated primary amines
(i.e., N-terminus and ε-NH<sub>2</sub> of lysine). Modified
arginine-terminated tryptic peptides have shown evidence of a covalent
modification at the N-terminus and a noncovalent interaction with
the arginine residue. The modified anions yield at least as much sequence
information upon CID as the unmodified cations for the small tryptic
peptides examined here and more sequence information for the large
tryptic peptides. This study represents the first demonstration of
gas-phase ion/ion reactions involving MALDI-derived ions. In this
case, covalent and electrostatic modification charge inversion is
shown to enhance MALDI tandem mass spectrometry of tryptic peptides
Electrospray Droplet Exposure to Organic Vapors: Metal Ion Removal from Proteins and Protein Complexes
The exposure of aqueous nanoelectrospray
droplets to various organic
vapors can dramatically reduce sodium adduction on protein ions in
positive ion mass spectra. Volatile alcohols, such as methanol, ethanol,
and isopropanol lead to a significant reduction in sodium ion adduction
but are not as effective as acetonitrile, acetone, and ethyl acetate.
Organic vapor exposure in the negative ion mode, on the other hand,
has essentially no effect on alkali ion adduction. Evidence is presented
to suggest that the mechanism by which organic vapor exposure reduces
alkali ion adduction in the positive mode involves the depletion of
alkali metal ions via ion evaporation of metal ions solvated with
organic molecules. The early generation of metal/organic cluster ions
during the droplet desolvation process results in fewer metal ions
available to condense on the protein ions formed via the charged residue
mechanism. These effects are demonstrated with holomyoglobin ions
to illustrate that the metal ion reduction takes place without detectable
protein denaturation, which might be revealed by heme loss or an increase
in charge state distribution. No evidence is observed for denaturation
with exposure to any of the organic vapors evaluated in this work
Affecting Protein Charge State Distributions in Nano-Electrospray Ionization via In-Spray Solution Mixing Using Theta Capillaries
Borosilicate
theta glass capillaries pulled to serve as nanoelectrospray
ionization emitters are used for short time-scale mixing of protein
and acid solutions during the electrospray process to alter protein
charge state distributions (CSDs) without modifying the sample solution.
The extent of protein CSD shifting/denaturing can be tailored by acid
identity and concentration. The observed CSD(s) are protein dependent,
and the short mixing time-scale enables the study of short-lived unfolding
intermediates and higher charge states of noncovalent protein complexes,
including those of holomyoglobin. Additionally, the theta tips provide
a simple and inexpensive method for mixing nonvolatile reagents such
as supercharging agents, which cannot be used with previously developed
vapor leak-in techniques, with protein solutions during the electrospray
process
Gas-Phase Conjugation to Arginine Residues in Polypeptide Ions via <i>N</i>-Hydroxysuccinimide Ester-Based Reagent Ions
Gas-phase conjugation to unprotonated arginine side-chains
via <i>N</i>-hydroxysuccinimide (NHS) esters is demonstrated
through
both charge reduction and charge inversion ion/ion reactions. The
unprotonated guanidino group of arginine can serve as a strong nucleophile,
resulting in the facile displacement of NHS from NHS esters with concomitant
covalent modification of the arginine residue. This reactivity is
analogous to that observed with unprotonated primary amines such as
the N-terminus or ε-amino group of lysine. In solution, however,
the arginine residues tend to be protonated at pH values low enough
to prevent hydrolysis of NHS esters, which would render them relatively
unreactive with NHS esters. This work demonstrates novel means for
gas-phase conjugation to arginine side chains in polypeptide ions
Selective Gas-Phase Oxidation and Localization of Alkylated Cysteine Residues in Polypeptide Ions via Ion/Ion Chemistry
The
thiol group in cysteine residues is susceptible to several
post-translational modifications (PTMs), including prenylation, nitrosylation,
palmitoylation, and the formation of disulfide bonds. Additionally,
cysteine residues involved in disulfide bonds are commonly reduced
and alkylated prior to mass spectrometric analysis. Several of these
cysteine modifications, specifically S-alkyl modifications, are susceptible
to gas-phase oxidation via selective ion/ion reactions with periodate
anions. Multiply protonated peptides containing modified cysteine
residues undergo complex formation upon ion/ion reaction with periodate
anions. Activation of the ion/ion complexes results in oxygen transfer
from the reagent to the modified sulfur residue to create a sulfoxide
functionality. Further activation of the sulfoxide derivative yields
abundant losses of the modification with the oxidized sulfur as a
sulfenic acid (namely, XSOH) to generate a dehydroalanine residue.
This loss immediately indicates the presence of an S-alkyl cysteine
residue, and the mass of the loss can be used to easily deduce the
type of modification. An additional step of activation can be used
to localize the modification to a specific residue within the peptide.
Selective cleavage to create c- and z-ions N-terminal to the dehydroalanine
residue is often noted. As these types of ions are not typically observed
upon collision-induced dissociation (CID), they can be used to immediately
indicate where in the peptide the PTM was originally located
Gas-Phase Transformation of Phosphatidylcholine Cations to Structurally Informative Anions via Ion/Ion Chemistry
Gas-phase transformation of synthetic
phosphatidylcholine (PC)
monocations to structurally informative anions is demonstrated via
ion/ion reactions with doubly deprotonated 1,4-phenylenedipropionic
acid (PDPA). Two synthetic PC isomers, 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphocholine (PC<sub>16:0/18:1</sub>) and
1-oleoyl-2-palmitoyl-<i>sn</i>-glycero-3-phosphocholine
(PC<sub>18:1/16:0</sub>), were subjected to this ion/ion chemistry.
The product of the ion/ion reaction is a negatively charged complex,
[PC + PDPA – H]<sup>−</sup>. Collisional activation
of the long-lived complex causes transfer of a proton and methyl cation
to PDPA, generating [PC – CH<sub>3</sub>]<sup>−</sup>. Subsequent collisional activation of the demethylated PC anions
produces abundant fatty acid carboxylate anions and low-abundance
acyl neutral losses as free acids and ketenes. Product ion spectra
of [PC – CH<sub>3</sub>]<sup>−</sup> suggest favorable
cleavage at the <i>sn</i>-2 position over the <i>sn</i>-1 due to distinct differences in the relative abundances. In contrast,
collisional activation of PC cations is absent of abundant fatty acid
chain-related product ions and typically indicates only the lipid
class via formation of the phosphocholine cation. A solution phase
method to produce the gas-phase adducted PC anion is also demonstrated.
Product ion spectra derived from the solution phase method are similar
to the results generated via ion/ion chemistry. This work demonstrates
a gas-phase means to increase structural characterization of phosphatidylcholines
via ion/ion chemistry
Fourier-Transform MS and Closed-Path Multireflection Time-of-Flight MS Using an Electrostatic Linear Ion Trap
An
electrostatic linear ion trap (ELIT) has been configured to
allow for the simultaneous acquisition of mass spectra via Fourier
transform (FT) techniques (frequency measurement) and via time-of-flight
(TOF; time measurement). In the former case, the time-domain image
charge derived from a pick-up electrode in the field-free region of
the ELIT is converted to frequency-domain data via Fourier transformation
(i.e., FT-ELIT MS). In the latter case, the time difference between
ion injection into the ELIT and ion detection after release from the
ELIT using a microchannel plate (MCP) enables the acquisition of multireflection
time-of-flight mass spectra (MR-TOF MS). The ELIT geometry facilitates
the acquisition of both types of data simultaneously because the detection
schemes are independent and do not preclude one another. The two MS
approaches exhibit a degree of complementarity. Resolution increases
much faster with time with the MR-TOF approach, for example, but the
closed-path nature of executing MR-TOF in an ELIT limits both the <i>m</i>/<i>z</i> range and the peak capacity. For this
reason, the FT-ELIT MS approach is most appropriate for wide <i>m</i>/<i>z</i> range applications, whereas MR-TOF
MS can provide advantages in a “zoom-in” mode in which
moderate resolution (<i>M</i>/Δ<i>M</i><sub>fwhm</sub> ≈ 10000) at short analysis times (10 ms) is desirable
Top-Down Interrogation of Chemically Modified Oligonucleotides by Negative Electron Transfer and Collision Induced Dissociation
Two
sets of synthetic 21–23mer oligonucleotides with various
types of 2′-position modifications have been studied with tandem
mass spectrometry using ion trap collision-induced dissociation (IT-CID)
and negative electron transfer (NET)-CID. A systematic study has been
conducted to define the limitations of IT-CID in sequencing such 2′-chemically
modified oligonucleotides. We found that IT-CID is sufficient in characterizing
oligonucleotide sequences that do not contain DNA residues, where
high sequence coverage can be achieved by performing IT-CID on multiple
charge states. However, oligonucleotides containing DNA residues gave
limited backbone fragmentation with IT-CID, largely due to dominant
fragmentation at the DNA residue sites. To overcome this limitation,
we employed the negative electron transfer to strip an electron from
the multiply charged oligonucleotide anion. Then, the radical anion
species formed in this reaction can fragment via an alternative radical-directed
dissociation mechanism. Unlike IT-CID, NET-CID mainly generates a
noncomplementary d/w ion series. Furthermore, we found that NET-CID
did not show preferential dissociations at the DNA residue sites and
thus generated higher sequence coverage for the studied oligonucleotide.
Information from NET-CID of different charge states is not fully redundant
such that the examination of multiple charge states can lead to more
extensive sequence confirmation. This work demonstrates that the NET-CID
is a valuable tool to provide high sequence coverage for chemically
modified oligonucleotides, and such detailed characterization can
serve as an important assay to control the quality of therapeutic
oligonucleotides that are produced under the good manufacture practice
(GMP) regulations
Gas-Phase Ion/Ion Reactions to Enable Radical-Directed Dissociation of Fatty Acid Ions: Application to Localization of Methyl Branching
Methyl
branching on the carbon chains of fatty acids and fatty
esters is among the structural variations encountered with fatty acids
and fatty esters. Branching in fatty acid/ester chains is particularly
prominent in bacterial species and, for example, in vernix caseosa
and sebum. The distinction of branched chains from isomeric straight-chain
species and the localization of branching can be challenging to determine
by mass spectrometry (MS). Condensed-phase derivatization strategies,
often used in conjunction with separations, are most commonly used
to address the identification and characterization of branched fatty
acids. In this work, a gas-phase ion/ion strategy is presented that
obviates condensed-phase derivatization and introduces a radical
site into fatty acid ions to facilitate radical-directed dissociation
(RDD). The gas-phase approach is also directly amenable to fatty acid
anions generated via collision-induced dissociation from lipid classes
that contain fatty esters. Specifically, divalent magnesium complexes
bound to two terpyridine ligands that each incorporate a ((2,2,6,6-tetramethyl-1-piperidine-1-yl)oxy)
(TEMPO) moiety are used to charge-invert fatty acid anions. Following
the facile loss of one of the ligands and the TEMPO group of the remaining
ligand, a radical site is introduced into the complex. Subsequent
collision-induced dissociation (CID) of the complex exhibits preferred
cleavages that localize the site(s) of branching. The approach is
illustrated with iso-, anteiso-,
and isoprenoid branched-chain fatty acids and an intact glycerophospholipid
and is applied to a mixture of branched- and straight-chain fatty
acids derived from Bacillus subtilis