2 research outputs found
Click Chemistry Facilitates Formation of Reporter Ions and Simplified Synthesis of Amine-Reactive Multiplexed Isobaric Tags for Protein Quantification
We report the development of novel reagents for cell-level
protein
quantification, referred to as Caltech isobaric tags (CITs), which
offer several advantages in comparison with other isobaric tags (e.g.,
iTRAQ and TMT). Click chemistry, copper(I)-catalyzed azide–alkyne
cycloaddition (CuAAC), is applied to generate a gas-phase cleavable
linker suitable for the formation of reporter ions. Upon collisional
activation, the 1,2,3-triazole ring constructed by CuAAC participates
in a nucleophilic displacement reaction forming a six-membered ring
and releasing a stable cationic reporter ion. To investigate its utility
in peptide mass spectrometry, the energetics of the observed fragmentation
pathway are examined by density functional theory. When this functional
group is covalently attached to a target peptide, it is found that
the nucleophilic displacement occurs in competition with formation
of b- and y-type backbone fragment ions regardless of the amino acid
side chains present in the parent bioconjugate, confirming that calculated
reaction energetics of reporter ion formation are similar to those
of backbone fragmentations. Based on these results, we apply this
selective fragmentation pathway for the development of CIT reagents.
For demonstration purposes, duplex CIT reagent is prepared using a
single isotope-coded precursor, allyl-<i>d</i><sub>5</sub>-bromide, with reporter ions appearing at <i>m</i>/<i>z</i> 164 and 169. Isotope-coded allyl azides for the construction
of the reporter ion group can be prepared from halogenated alkyl groups
which are also employed for the mass balance group via <i>N</i>-alkylation,
reducing the cost and effort for synthesis of isobaric pairs. Owing
to their modular designs, an unlimited number of isobaric combinations
of CIT reagents are, in principle, possible. The reporter ion mass
can be easily tuned to avoid overlapping with common peptide MS/MS
fragments as well as the low mass cutoff problems inherent in ion
trap mass spectrometers. The applicability of the CIT reagent is tested
with several model systems involving protein mixtures and cellular
systems
Designer Reagents for Mass Spectrometry-Based Proteomics: Clickable Cross-Linkers for Elucidation of Protein Structures and Interactions
We present novel homobifunctional amine-reactive clickable
cross-linkers
(CXLs) for investigation of three-dimensional protein structures and
protein–protein interactions (PPIs). CXLs afford consolidated
advantages not previously available in a simple cross-linker, including
(1) their small size and cationic nature at physiological pH, resulting
in good water solubility and cell-permeability, (2) an alkyne group
for bio-orthogonal conjugation to affinity tags via the click reaction
for enrichment of cross-linked peptides, (3) a nucleophilic displacement
reaction involving the 1,2,3-triazole ring formed in the click reaction,
yielding a lock-mass reporter ion for only clicked peptides, and (4)
higher charge states of cross-linked peptides in the gas-phase for
augmented electron transfer dissociation (ETD) yields. Ubiquitin,
a lysine-abundant protein, is used as a model system to demonstrate
structural studies using CXLs. To validate the sensitivity of our
approach, biotin-azide labeling and subsequent enrichment of cross-linked
peptides are performed for cross-linked ubiquitin digests mixed with
yeast cell lysates. Cross-linked peptides are detected and identified
by collision induced dissociation (CID) and ETD with linear quadrupole
ion trap (LTQ)-Fourier transform ion cyclotron resonance (FTICR) and
LTQ-Orbitrap mass spectrometers. The application of CXLs to more complex
systems (e.g., in vivo cross-linking) is illustrated by Western blot
detection of Cul1 complexes including known binders, Cand1 and Skp2,
in HEK 293 cells, confirming good water solubility and cell-permeability