Protein Mass Spectrometry Laboratory

Key personnel:
mgr Michał Tracz

Scientific advisor:
prof. dr hab. Artur Krężel

Laboratory is oriented towards providing a complex scientific service related to protein mass spectrometry for research projects carried out at the Faculty of Biotechnology. Scientific cooperation with outside parties is also possible. Details on our equipment and current capabilities can be found below.

For analysis inquiries and consulting please contact: michal.tracz2@uwr.edu.pl

Equipment

LC-MS equipment:

• MS:
  Waters Synapt XS HRMS (ESI Q-IMS-TOF)
  ion sources:
  Zspray LockSpray II (µL flow rate applications)
  Zspray NanoLockSpray (nL flow rate and offline applications)
• LC:
  Waters Acquity NanoUPLC M-Class
  optionally equipped with the HDX Manager module (for HDX applications)

Licensed Software Packages:

MassLynx, PLGS (Protein Lynx Global Server)*, Progenesis QI for Proteomics*, DynamX, Mascott

^{*PLGS and Progenesis QIP are the vendor recommended software packages especially dedicated for Waters instrument-generated raw data formats of bottom-up analyses}

Open-source software:

SkyLine, ProSight Lite

The laboratory is equipped with basic labware required for LC-MS sample preparation.

The purchase of the LC-MS equipment was financially supported by the Research Equipment Fund (FAB) of the “Excellence Initiative — Research University” (IDUB) program for the University of Wroclaw.

Analysis scope

Routinely executed non-proteomic analyses:

• intact protein mass measurements (via C4 RP-LC)
• intact protein top-down analyses (via C4 RP-LC and CID or ETD fragmentation) for modification or PTM analysis on a pure protein

Specially addressed non-proteomic analyses:

• targeted protein quantification from biological and non-biological matrices via ToF-MRM
• protein or peptide hydrogen-deuterium exchange (HDX) structural analyses
• protein MS in non-denaturing conditions (native MS)

Routinely executed proteomic analyses:

• bottom-up protein identification* from biological matrices, PAGE gel matrices (including SDS-PAGE gels) and CoIP (pull-down) resins
• bottom-up protein IBAQ quantification** from biological matrices, PAGE gel matrices (including SDS-PAGE gels) and CoIP (pull-down) resins
  ^{*The standard bottom-up workflow includes a one-enzyme proteolytic cleavage by trypsin, and a peptide desalting step prior to sample’s placement on the system.
  **IBAQ quantification includes a spike of an internal signal standard}

Specially addressed proteomic analyses:

• in-depth bottom-up protein IBAQ quantification from biological matrices; for a significantly larger protein hit count, than the one obtainable from a standard analysis of the given sample
• bottom-up identification of protein PTMs and/or modifications from biological matrices

Expertise

List of scientific advisor’s selected research works encompassing protein MS:

1. Structural Characterization of Cu(I)/Zn(II)-metallothionein-3 by Ion Mobility Mass Spectrometry and Top-Down Mass Spectrometry
2. Ion mobility mass spectrometry and molecular dynamics simulations unravel the conformational stability of zinc metallothionein-2 species
3. An Integrated Mass Spectrometry and Molecular Dynamics Simulations Approach Reveals the Spatial Organization Impact of Metal-Binding Sites on the Stability of Metal-Depleted Metallothionein-2 Species
4. Mass Spectrometry-Based Structural Analysis of Cysteine-Rich Metal-Binding Sites in Proteins with MetaOdysseus R Software
5. Metal- and Affinity-Specific Dual Labeling of Cysteine-Rich Proteins for Identification of Metal-Binding Sites
6. Crosstalk of the structural and zinc buffering properties of mammalian metallothionein-2

Key personnel experience

• 07/2022: Internship at the MS core facility of the Makrokomplex consortium, University of South Bohemia, Czech Republic
• 01/2023: 10-day Waters applicatory on-site training course delivered by Waters applicatory specialist (dr. Alex Muck)