Peptide facility

Peptides are routinely synthesized in the range of 5-80 amino acids, in an amount of 1-200 µmol (smaller and larger amounts are also possible). All peptides are analyzed by LC-MS and if needed purified by RP-HPLC. For analytical purposes the peptide core facility has a Waters liquid chromatography electro spray mass spectrometer at its disposal. More than 6000 peptides are synthesized on a yearly basis.

Services

We routinely use Fmoc-solid phase chemistry and have 2 Syro II robot synthesizers and a Intavis synthesizer equipped with real time Fmoc-monitoring. On one of the Syro's 48 peptides can be synthesized simultaneously on a typical 25 µmol scale. The other Syro can synthesize 6 X 96 peptides in up to 4 µmol amounts in one run for screening purposes. The Intavis synthesizer is used for synthesizing larger amounts of peptides and for fine-tuning difficult sequences using the Fmoc-monitoring methodology.

 Besides "straight forward" peptides it is also possible to synthesize specifically modified peptides.

  • Ubiquitin(like) and derivatives thereof
  • Variations at the N-terminus; biotinylation (w/wo a spacer), acetylation, fluorophores etc.Phosphorylated peptides
  • Peptides with a side chain modification (such as thiolysine e.g.)
  • UV cleavable peptides
  • Unnatural amino acid incorporation
  • Alanine scans

Equipment

Being both an organic chemistry lab and a biochemistry/chemical biology lab working with reagents ranging from in house prepared small molecule inhibitor libraries all the way up to activity based probes consisting of modified di-ubiquitin proteins, we routinely use a wide variety of equipment:

Peptide synthesizers

  • MultiSynTech Syro II - 1 x 24 parallel synthesizer (each 40 umol)
  • MultiSynTech Syro II - 6 x 96 parallel synthesizer (each 4 umol)
  • Intavis MultiPep CF 

Mass Spectrometers

  • WATERS Alliance  LCMS coupled to a LCT-premier Mass Spectrometer
  • WATERS Acquity H-class UPLC coupled to a high resolution XEVO G2S-XTOF Mass Spectrometer 

HPLC-systems

  • WATERS 2545 LCMS coupled to a 3100 Mass Spectrometer; mass triggered collection during HPLC   purification mostly used for small molecules.
  • WATERS 2535 LCMS coupled to a 2545 UV-VIS detector; routinely used for large scale purifications of Ubl's 
  • WATERS 1525 EF LCMS coupled to a 2487 dual labda Absorbance Detector; routinely used for peptide purifications
  • Shimadzu LC-20 LCMS system; routinely used for small scale purifications of peptides and Ubl's

FPLC-systems

  • 2x BUCHI-C620 automated colomn set-ups for straight phase silica separations
  • BIORAD NGC system for cat-/anion exchange and size exclusion gelfiltration

NMR-system

  • Bruker Ultrashield 300 MHz


CONTACT

Cami Talavera Ormeno
E-mail: peptides@lumc.nl
Tel: +31 (0) 71 526 8730

Peptide Facility
Department of Cell and Chemical Biology (CCB)
Leiden University Medical Center (LUMC)

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