4 research outputs found
Corona Discharge Suppression in Negative Ion Mode Nanoelectrospray Ionization via Trifluoroethanol Addition
Negative ion mode
nanoelectrospray ionization (nESI) is often utilized
to analyze acidic compounds, from small molecules to proteins, with
mass spectrometry (MS). Under high aqueous solvent conditions, corona
discharge is commonly observed at emitter tips, resulting in low ion
abundances and reduced nESI needle lifetimes. We have successfully
reduced corona discharge in negative ion mode by trace addition of
trifluoroethanol (TFE) to aqueous samples. The addition of as little
as 0.2% TFE increases aqueous spray stability not only in nESI direct
infusion, but also in nanoflow liquid chromatography (nLC)/MS experiments.
Negative ion mode spray stability with 0.2% TFE is approximately 6Ă—
higher than for strictly aqueous samples. Upon addition of 0.2% TFE
to the mobile phase of nLC/MS experiments, tryptic peptide identifications
increased from 93 to 111 peptides, resulting in an average protein
sequence coverage increase of 18%
Characterization of <i>O</i>-Sulfopeptides by Negative Ion Mode Tandem Mass Spectrometry: Superior Performance of Negative Ion Electron Capture Dissociation
Positive ion mode collision-activated dissociation tandem
mass
spectrometry (CAD MS/MS) of <i>O</i>-sulfopeptides precludes
determination of sulfonated sites due to facile proton-driven loss
of the highly labile sulfonate groups. A previously proposed method
for localizing peptide and protein <i>O</i>-sulfonation
involves derivatization of nonsulfonated tyrosines followed by positive
ion CAD MS/MS of the corresponding modified sulfopeptides for diagnostic
sulfonate loss. This indirect method relies upon specific and complete
derivatization of nonsulfonated tyrosines. Alternative MS/MS activation
methods, including positive ion metastable atom-activated dissociation
(MAD) and metal-assisted electron transfer dissociation (ETD) or electron
capture dissociation (ECD) provide varying degrees of sulfonate retention.
Sulfonate retention has also been reported following negative ion
MAD and electron detachment dissociation (EDD), which also operates
in negative ion mode in which sulfonate groups are less labile than
in positive ion mode. However, an MS/MS activation technique that
can effectively preserve sulfonate groups while providing extensive
backbone fragmentation (translating to sequence information, including
sulfonated sites) with little to no noninformative small molecule
neutral loss has not previously been realized. Here, we report that
negative ion CAD, EDD, and negative ETD (NETD) result in sulfonate
retention mainly at higher charge states with varying degrees of fragmentation
efficiency and sequence coverage. Similar to previous observations
from CAD of sulfonated glycosaminoglycan anions, higher charge states
translate to a higher probability of deprotonation at the sulfonate
groups thus yielding charge-localized fragmentation without loss of
the sulfonate groups. However, consequently, higher sulfonate retention
comes at the price of lower sequence coverage in negative ion CAD.
Fragmentation efficiency/sequence coverage averaged 19/6% and 33/20%
in EDD and NETD, respectively, both of which are only applicable to
multiply-charged anions. In contrast, the recently introduced negative
ion ECD showed an average fragmentation efficiency of 69% and an average
sequence coverage of 82% with complete sulfonate retention from singly-
and doubly-deprotonated sulfopeptide anions
Targeted Annotation of S‑Sulfonylated Peptides by Selective Infrared Multiphoton Dissociation Mass Spectrometry
Protein
S-sulfinylation (R–SO<sub>2</sub><sup>–</sup>) and S-sulfonylation
(R–SO<sub>3</sub><sup>–</sup>) are irreversible oxidative
post-translational modifications of
cysteine residues. Greater than 5% of cysteines are reported to occupy
these higher oxidation states, which effectively inactivate the corresponding
thiols and alter the electronic and physical properties of modified
proteins. Such higher oxidation states are reached after excessive
exposure to cellular oxidants, and accumulate across different disease
states. Despite widespread and functionally relevant cysteine oxidation
across the proteome, there are currently no robust methods to profile
higher order cysteine oxidation. Traditional data-dependent liquid
chromatography/tandem mass spectrometry (LC/MS/MS) methods generally
miss low-occupancy modifications in complex analyses. Here, we present
a data-independent acquisition (DIA) LC/MS-based approach, leveraging
the high IR absorbance of sulfoxides at 10.6 ÎĽm, for selective
dissociation and discovery of S-sulfonated peptides. Across peptide
standards and protein digests, we demonstrate selective infrared multiphoton
dissociation (IRMPD) of S-sulfonated peptides in the background of
unmodified peptides. This selective DIA IRMPD LC/MS-based approach
allows identification and annotation of S-sulfonated peptides across
complex mixtures while providing sufficient sequence information to
localize the modification site
Free Radical Initiated Peptide Sequencing for Direct Site Localization of Sulfation and Phosphorylation with Negative Ion Mode Mass Spectrometry
Tandem
mass spectrometry (MS/MS) is the primary method for discovering,
identifying, and localizing post-translational modifications (PTMs)
in proteins. However, conventional positive ion mode collision induced
dissociation (CID)-based MS/MS often fails to yield site-specific
information for labile and acidic modifications due to low ionization
efficiency in positive ion mode and/or preferential PTM loss. While
a number of alternative methods have been developed to address this
issue, most require specialized instrumentation or indirect detection.
In this work, we present an amine-reactive TEMPO-based free radical
initiated peptide sequencing (FRIPS) approach for negative ion mode
analysis of phosphorylated and sulfated peptides. FRIPS-based fragmentation
generates sequence informative ions for both phosphorylated and sulfated
peptides with no significant PTM loss. Furthermore, FRIPS is compared
to positive ion mode CID, electron transfer dissociation (ETD), as
well as negative ion mode electron capture dissociation (niECD) and
CID, both in terms of sequence coverage and fragmentation efficiency
for phospho- and sulfo-peptides. Because FRIPS-based fragmentation
has no particular instrumentation requirements and shows limited PTM
loss, we propose this approach as a promising alternative to current
techniques for analysis of labile and acidic PTMs