Mass spectrometry

· Expertise and mass spectrometry facility in proteomics

Protein identification from 1D/2D electrophoresis gel or from complex mixtures cellular extracts, posttranslational modifications characterization, quantitative approaches(targeted or not), study of non-covalent complexes

· Services : simple analysis, medium and long term collaboration works, researchesand implementation of new methodologies: for academics (UPMC, universities/CNRS/INSERM) and private compagnies.

The main goal of the platform is to offer instrumentation and expertise in mass spectrometry for protein identification and characterization in collaboration works especially at UPMC, in routine analysis and research. Three people make up our group, before recruiting in 2014 a Research Engineer: G Clodic (Assistant Engineer-UPMC), E Sachon (Assistant-Professor UPMC) and G Bolbach (DR-CNRS). Training and teaching are also activities of the platform.

Four mass spectrometers are available and allow implementing all general studies and analysis in proteomics. They are based on complementary MALDI and ESI ionization and various analyzers (TOF, Quadrupole and Orbitrap). They perform accurate mass measurement in MS and sequence information can be gained using tandem mass spectrometry (MS/MS) by fragmenting selected ions. For complex mixtures of peptides on-line coupling with liquid chromatography is frequently used. All the instruments are connected to protein data banks through search engine such as Mascot and Sequest.

Identification of small biomolecules (oligonucleotides, steroids…) can also be addressed.

Most of the routine analysis and collaborations are developed at UPMC, in public institutes and research centers ( universities, CNRS, INSERM, INRA) and with private companies.

Material

Material:

MALDI-TOF (DE-Pro, ABSciex) :fitted with a nitrogen laser (l=337 nm, 20 Hz) The time-of-flight analyzer can be used in linear mode for high mass range (m/z>1000) and a reflector mode for medium mass range (m/z 500- 6000) and high resolution. It is mainly used in self-service by trained people. The same targets can be also analyzed in MS/MS mode on the MALDI-TOF-TOF.

MALDI-TOF-TOF (4700 Proteomic Analyzer, ABSciex): fitted with NdYAG laser (l=355 nm, 200 Hz). In linear mode, a high mass detector (CovalX) allows the detection of high mass proteins (Mw up to 106 Da). In reflector mode, high resolution (R~20 000) is achieved in the range m/z<6000 (peptide mass fingerprint) for a moderate number of peptides. A mode MS/MS with a collision cell (CID with high collision energy 1 keV) allows fragmentation of selected ions to get sequence information (protein identification, post-translationnal modifications, natural or synthetic peptides…). An off-line coupling with nanoLC can be implemented by means of an automatic spotter (ProBot, Dionex) using the MALDI sample plate.

ESI-Qtrap (QTRAP LC/MS/MS System, ABSciex) : this instrument is a hybrid triple quadrupole linear ion (range m/z < 1700). It can be coupled on line with a liquid chromatography device (Ultimate 3000 Dionex) using medium flow rate (µL/min). This mass spectrometer is mainly dedicated to the analysis of small molecules (steroids, oligonucleotides….) in complex mixtures. MS and MS/MS modes are used in routine analysis. MRM (Multiple Reaction Monitoring) is also a very useful mode allowing the detection and quantitation of specific compounds knowing their mass and that of fragments.

ESI-Orbitrap (LTQ-Orbitrap XL+ ETD, ThermoFisher Scientific) :this hybrid mass spectrometer incorporates a linear ion trap (LTQ) and the Orbitrap analyzer (range m/z <2000 in routine analysis). This last one offers high resolution (R ~100 000), very accurate (~ppm) mass measurement and high dynamic range. Nanospray ion sources are used in a static or dynamic mode when coupled to nanoLC (a few tens of nL/min). This last mode is the most appropriate one in proteomics for identification and characterization of proteins from complex mixtures (digests of cellular extracts….). Exact mass measurement for low mass compounds (m/z<400) is possible.

Three complementary fragmentation modes are available: CID low energy collision, HCD high energy collision and ETD dissociation induced by electron transfer.

All these mass spectrometers offer complementary performances (ionization, fragmentation, robustness, resolution, precision, easy to handle sample…). All are connected to data banks (NCBI, SwissProt…) through search engines (Mascot, Sequest…).

Services

Presentation of the offers

Discussion with the applicants are wished to determine the best analytical strategy and appropriate instrumentation. Scientific support can be provided on the data interpretation.

Type of offers :

MALDI-TOF mass spectrometer in self-service: after a 2 days training (theory and practice) for researchers, engineers, technicians as well as M2 and PhD students and post-docs. Sample preparation, data acquisition and processing are under the responsibility of the applicant. The scientific and technical support is provided by the members of the platform.

Routine analysis and collaboration: only the members of the platform can use the mass spectrometers and coupling devices. However when requested by the applicant this latter can follow the MS experiment and measurement as well as data processing.

Mass spectra are sent to the applicant as pdf files. The connection of USB keys in the computers (acquisition and processing) of the platform is completely forbidden.

Long-term collaborative project: applicants can participate to the experiments and also to the data processing for protein identification in data banks.

Methodologies

  • Protein identification: MS and MS/MS protein identification from digests of 1D or 2D gels, from complex cellular extract. Trypsine digestion can be performed at the platform.Analysis: nanoLC ESI-LTQ-Orbitrap and or MALDI-TOF-TOF.
  • Characterization of postranslational modifications: especially, enrichment and purification of phosphopeptides in phosphoprotein studies.
  • Quantification: ITRAQ and SILAC, internal standard, dimethylation….
  • MRM (or SRM)analysis: quantitation and detection of low mass compounds in very complex mixtures (ESI-Qtrap).
  • Interactomics: crosslinking and photolabeling techniques for the study of protein complexes.
  • Specific methodologies can be developed and implemented in collaborative works