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Protein Chemistry Facility core facility

The protein chemistry facility (PCF) is a research and service platform of IBAR performing protein, peptide, proteome and subproteome analysis.

The protein chemistry facility (PCF) is a research and service platform of IBAR performing protein, peptide, proteome and subproteome analysis. The facility was established in 2012 by a EU funded grant PROCERA (POSCCE-A2-O2.2.1-2009-4) in the Department of Bioinformatics and Structural Biochemistry (IBAR-DBSB).

Its main research topics are closely related with the focus-area of other groups and departments of IBAR: proteome and subproteome characterization in melanoma, protein-protein interactions in biological systems, co- and post-translational modification analysis with a special focus on glycosylation, imunopeptidome investigation, identification of specific protein interactors. Another priority is the study of protein phosphorylation, a dynamic process that implies the complementary activities of protein kinases and protein phosphatases. Mass spectrometric analysis of the samples implies the use of CID, HCD and ETD fragmentation methods. Data analysis and interpretation of results is made using Thermo Scientific™ Proteome Discoverer™ software. For phosphosite localization, besides the automated analysis (PhosphoRS Node in Proteome Discoverer) a manual inspection of the raw tandem mass spectra of the peptide is often made.

As a service unit the facility can provide the following methods of analysis:

  • analytical and preparative HPLC of small molecules and biomolecules
  • nanoHPLC separation of peptides
  • low-/high-resolution mass spectrometry (MS) and tandem MS
  • sample preparation of proteins and peptides for LC/MS analysis (spectrophotometric -UV-VIS, fluorimetric and electrophoretic methods -SDS-PAGE)
  • bioinformatic MS data analysis
  • batch or column fast protein chromatography

Thermo Scientific Easy-nanoLC II:

The Easy-nanoLC is a binary pump system able to separate biomolecules on capillary columns at low flows (100-1000 nL/min). It is used to separate peptides on reversed-phase C18 nano columns. 

 

Thermo Scientific LTQ-Orbitrap Velos Pro:

This is a hybrid mass analyzer composed of a fast linear ion trap (up to 12.5 Hz) and a high resolution Orbitrap analyzer (up to 100 000 resolution at m/z 400) with mass accuracy in the low ppm range. It is used for peptide, protein, small molecule and other biomolecules analysis.

 

Thermo Scientific Accela 600:

This separation instrument is equipped with a 600 bar pump allowing small molecule and biomolecule routine analysis, but also applications requiring high flow separations (up to 5 ml/min). It is connected to two detection systems: an UV-VIS PDA detector and a fluorescence detector (Thermo Finnigan Surveyor FL Plus Detector). It is used for peptide fractionation and purification.

 

Thermo Scientific Dionex IC5000:

Dionex IC5000 is an instrument dedicated to ion chromatography. It features a quaternary pump, an electrochemical detector and an additional UV detector (also includes a desalting device) for to the analysis of various ions. It is dedicated to the to the separation highly polar molecules, like amino acids, glycans, hydrogen peroxide or nitrates.

As a research platform the members of the facility actively participate in the elaboration and optimization of new methods and protocols for protein analysis in biological systems. Furthermore the team also contributes to the elaboration of grant applications and research publications that include the expertise of its members.

Selected research grants that included the participation of IBAR-PCF members:

  • TYRPRES, Romanian-Swiss Project IZERZ0_142216, 2012–2015 "Role of the ERAD pathway in the degradation of tyrosinase and production of antigenic peptides in human melanoma"
  • LYSOMOD_734825 2017-2021 – “Genetic and Small Molecule Modifiers of Lysosomal Function” - International Collaboration IMUNOPEP 2016-2020, Mass spectrometry based investigation of the oxidative stress as a potential key-player in the immunobiology of melanoma (PN-III-P1-1.1-PD-2016-1528)
  • PRODEGRAD 2013-2016 -"Selection of protein degradative pathways in the pathogenesis of human diseases (PN-II-ID-PCE-2012-4-0350) MOLINT 2011-2016 - "Modeling molecular complexes and assemblies with experimental and bioinformatic constraints" (PN-II-ID-PCE-2011-3-0342

Selected publications that included the participation of IBAR-PCF members:

  • Chirițoiu GN#, Jandus C#, Munteanu CVA#, Ghenea S, Gannon PO, Romero P, Petrescu SM, “Epitope located N-glycans impair the MHC-I epitope generation and presentation", Electrophoresis; 37(11): 1448-60, 2016 DOI: [10.1002/elps.201500449]
  • Sârbu M, Munteanu CVA, Dehelean L, Petrescu AJ, Peter-Katalinic J, Zamfir AD, "Identification and structural characterization of novel O- and N-glycoforms in the urine of a Schindler disease patient by Orbitrap mass spectrometry", Journal of Mass Spectrometry; 50(9): 1044-56, 2015 DOI: [10.1002/jms.3616]
  • Petrareanu G, Balasu MC, Vacaru AM, Munteanu CVA, Ionescu AE, Matei I, Szedlacsek SE, "Phosphoketolases from Lactococcus lactis, Leuconostoc mesenteroides and Pseudomonas aeruginosa: Dissimilar sequences, similar substrates but distinct enzymatic characteristics", Applied Microbiology and Biotechnology; 98(18): 7855-67, 2014 DOI: [10.1007/s00253-014-5723-6]
  • Tănase CI, Drăghici C, Shova S., Hanganu A., Gal E., Munteanu CVA., A long-range tautomeric effect on a new Schiff isoniazid analogue, evidenced by NMR study and X-ray crystallography, New Journal of Chemistry 42(17): 14459-14468, 2018 DOI:[ 10.1039/c8nj01680a]