Project description:UVPD was implemented on an Orbitrap Q-Exactive plus equipped with a ESI/EP-MALDI. UVPD of MALDI generated ions was benchmarked against MALDI-ISD, MALDI-HCD and ESI-UVPD. MALDI UVPD outperformed MALDI HCD and ISD efficiently sequencing small proteins ions.
Project description:Crosslinking mass spectrometry (XL-MS) has become a key technique for investigating protein-protein interactions and the architecture of biological systems. This study compares the Orbitrap Astral and Eclipse mass spectrometers in XL-MS workflows, emphasizing their performance in detecting low-abundance crosslinked peptides. Using Cas9 crosslinked with PhoX and DSSO, the Astral demonstrated over 40% higher crosslink identifications due to its superior ion transmission and sensitivity, enabled by its multi-reflection time-of-flight analyzer. Single higher-energy collisional dissociation (HCD) fragmentation was more effective than stepped HCD on the Astral, while the Eclipse showed similar results for both methods. Gradient optimization revealed that extended separation times improved crosslink identifications. These findings highlight the critical role of instrumentation and workflow optimization in advancing XL-MS for structural proteomics.
Project description:The project aimed to identify interaction sites of RNF168 with the Nucleosomes. RNF168 interaction with the Nucleosome was probed by BS3 crosslinking to support structure modeling based on NMR and mutagenesis experiments
Project description:In this study we compared three different fragmentation techniques and two combined fragmentation schemes available on a novel tribrid mass spectrometer (Orbitrap Fusion Lumos, Themro Fisher Scientific) CID, HCD, ETD, ETD with supplemental CID (ETciD) and ETD with supplemental HCD (EThcD) on cross-linked peptides obtained by tryptic cleavage of SDA-cross-linked Human Serum Albumin (HSA). The three-dimensional structure of HSA has been resolved by X-ray crystallography [35] and is used as ground-truth to evaluate the identification results. Right choice of the fragmentation method allows increasing the number of identified linkage sites, increasing the sequence coverage of both linked peptides thereby reducing the second peptide problem, and increasing the precision of cross-link site calling.
Project description:BS3 Crosslinking experiments were performed on in vitro preparations of histone methyltransferase dimers. Each sample was fractionated into two size-exclusion chromatography fractions that were acquired by alternating HCD/EThcD scans of the same precursor on a Fusion-Lumos instrument.
File mappings:
GLP homodimer, SEC1: Z20210712-08
GLP homodimer, SEC2: Z20210712-09
G9a homodimer, SEC1: Z20210712-10
GLP homodimer, SEC2: Z20210712-11
G9aGLP heterodimer, SEC1: Z20210712-12
G9aGLP heterodimer, SEC2: Z20210712-13
Project description:The data sets of human IgG and human fibrinogen are used as test data for the publication of glyXtoolMS (https://github.com/glyXera/glyXtoolMS). GlyXtoolMS is an open-source analysis software for the (semi)automated targeted analysis of glycopeptide mass spectrometry data using OpenMS/TOPPAS as a framework and pipeline engine. The proteins were selected to show the successful analysis of both N-glycopeptide and O-glycopeptide samples. The samples were measured by nano reversed phase liquid chromatography coupled online to an electrospray ionization orbitrap mass spectrometer (nano RP-LC ESI- OT-MS/MS; LTQ Orbitrap Elite, Thermo Scientific, Waltham, MA, USA) with HCD fragmentation.