20 research outputs found
Exploiting reversible interactions: hydrogels and protein cross-linkers
A series of low molecular weight thermoreversible cystine hydrogelators were
synthesised via solid-phase chemistry. Novel hydrogels were found to gelate at
concentrations of <2 mM using microwave super-heating. Benzoyl cystine amide
derivative hydrogel, which could form at a concentration of 0.5 mM, equivalent to
0.022% w/w of gelator with respect to water (an incredible 111,000 molecules of
water gelated per single molecule of gelator), was applied to cell culture of cervical
cancer (HeLa) cells, which were found to distribute within the gel. Hydrogels were
produced on a microarray format using a novel strategy involving deposition of
hydrogel solutions by inkjet printing. The incorporation of fluorescent dye
(Rhodamine B) into hydrogels provided a novel means for studying hydrogel
morphology.Reversible boronate chemistry was implemented for the capture and release of
proteins and peptides onto a solid-support as part of a modified peptide enrichment
strategy. The strategy was proven following synthesis of hydroxamic acid and
catechol modified peptides and a study of their interaction with solid-supported
phenylboronic acid. NHS active ester affinity tags and cross-linkers were synthesised
and applied to a 3D proteomics cross-linking analysis pipe-line. The introduction of a
PEG unit led to a cross-linker with increased hydrophilicity and improved
observation of both inter and intra-protein cross-links by mass spectrometry
Light-Induced Orthogonal Fragmentation of Crosslinked Peptides
Crosslinking mass spectrometry provides pivotal information on the structure and interaction of proteins. MS-cleavable crosslinkers are regarded as a cornerstone for the analysis of complex mixtures. Yet they fragment under similar conditions as peptides, leading to mixed fragmentation spectra of the crosslinker and peptide. This hampers selecting individual peptides for their independent identification. Here, we introduce orthogonal cleavage using ultraviolet photodissociation (UVPD) to increase crosslinker over peptide fragmentation. We designed and synthesized a crosslinker that can be cleaved at 213 nm in a commercial mass spectrometer configuration. In an analysis of crosslinked Escherichia coli lysate, the crosslinker-to-peptide fragment intensity ratio increases from nearly 1 for a conventionally cleavable crosslinker to 5 for the UVPD-cleavable crosslinker. This largely increased the sensitivity of selecting the individual peptides for MS3, even more so with an improved doublet detection algorithm. Data are available via ProteomeXchange with identifier PXD040267.<br/
Dual-bioorthogonal catalysis by a palladium peptide complex
Artificial metalloenzymes (ArMs) enrich bioorthogonal
chemistry
with new-to-nature reactions while limiting metal deactivation and
toxicity. This enables biomedical applications such as activating
therapeutics in situ. However, while combination
therapies are becoming widespread anticancer treatments, dual catalysis
by ArMs has not yet been shown. We present a heptapeptidic ArM with
a novel peptide ligand carrying a methyl salicylate palladium complex.
We observed that the peptide scaffold reduces metal toxicity while
protecting the metal from deactivation by cellular components. Importantly,
the peptide also improves catalysis, suggesting involvement in the
catalytic reaction mechanism. Our work shows how a palladium-peptide
homogeneous catalyst can simultaneously mediate two types of chemistry
to synthesize anticancer drugs in human cells. Methyl salicylate palladium
LLEYLKR peptide (2-Pd) succeeded to simultaneously produce
paclitaxel by depropargylation, and linifanib by Suzuki–Miyaura
cross-coupling in cell culture, thereby achieving combination therapy
on non-small-cell lung cancer (NSCLC) A549 cells
Optimized fragmentation regime for diazirine photo-cross-linked peptides
Cross-linking/mass spectrometry has
evolved into a robust technology
that reveals structural insights into proteins and protein complexes.
We leverage a new tribrid instrument with improved fragmentation capacities
in a systematic comparison to identify which fragmentation method
would be best for the identification of cross-linked peptides. Specifically,
we explored three fragmentation methods and two combinations: collision-induced
dissociation (CID), beam-type CID (HCD), electron-transfer dissociation
(ETD), ETciD, and EThcD. Trypsin-digested, SDA-cross-linked human
serum albumin (HSA) served as a test sample, yielding over all methods
and in triplicate analysis in total 2602 matched PSMs and 1390 linked
residue pairs at 5% false discovery rate, as confirmed by the crystal
structure. HCD wins in number of matched peptide-spectrum-matches
(958 PSMs) and identified links (446). CID is most complementary,
increasing the number of identified links by 13% (58 links). HCD wins
together with EThcD in cross-link site calling precision, with approximately
62% of sites having adjacent backbone cleavages that unambiguously
locate the link in both peptides, without assuming any cross-linker
preference for amino acids. Overall quality of spectra, as judged
by sequence coverage of both peptides, is best for EThcD for the majority
of peptides. Sequence coverage might be of particular importance for
complex samples, for which we propose a data dependent decision tree,
else HCD is the method of choice. The mass spectrometric raw data
has been deposited in PRIDE (PXD003737)
Light-Induced Orthogonal Fragmentation of Crosslinked Peptides
Crosslinking mass spectrometry provides pivotal information on the structure and interaction of proteins. MS-cleavable crosslinkers are regarded as a cornerstone for the analysis of complex mixtures. Yet they fragment under similar conditions as peptides, leading to mixed fragmentation spectra of the crosslinker and peptide. This hampers selecting individual peptides for their independent identification. Here, we introduce orthogonal cleavage using ultraviolet photodissociation (UVPD) to increase crosslinker over peptide fragmentation. We designed and synthesized a crosslinker that can be cleaved at 213 nm in a commercial mass spectrometer configuration. In an analysis of crosslinked Escherichia coli lysate, the crosslinker-to-peptide fragment intensity ratio increases from nearly 1 for a conventionally cleavable crosslinker to 5 for the UVPD-cleavable crosslinker. This largely increased the sensitivity of selecting the individual peptides for MS3, even more so with an improved doublet detection algorithm. Data are available via ProteomeXchange with identifier PXD040267.<br/
Blind Evaluation of Hybrid Protein Structure Analysis Methods based on Cross-Linking
Hybrid methods combine experimental data and computational modeling to analyze protein structures that are elusive to structure determination. To spur the development of hybrid methods, we propose to test them in the context of the CASP experiment and would like to invite experimental groups to participate in this initiative