Biopolymers & Proteomics Laboratory

The David H.Koch Institute for Integrative Cancer Research at MIT

Bldg 76 Room 181

Telephone: 617-253-7038

Cellulose Peptide Array (Membrane) 20nmol

Description of ABIMED Peptide Arrayer

The ABIMED peptide arrayer system consists of a computer controlled Gilson diluter and XYZ liquid handling robot which allows the deposition on amino-PEG cellulose membranes of individual activated amino acids resulting in peptide formation using FMOC chemistry. Peptides are synthesized with a theoretical limit of 40 residues although the manufacturer recommends 20 residues as a more reasonable limit. Our mass spectrometry of peptides cleaved from the membranes indicates that a maximum length of 12 residues ensures that there will be some material covalently linked to each spot with the correct molecular weight.

Up to four 9 x 13 cm membranes, which can be reused 10-20 times, can be synthesized simultaneously. Each membrane can hold 384 peptide spots. Each spot consists of 20 nmol starting peptide. Basically, the membranes are loaded onto the XYZ robot and 20 nmol of activated FMOC beta alanine are deposited in a 200 nanoliter volume forming a spot with a diameter of ca. 3 mm. After coupling, the membrane is then acetylated to prevent unwanted synthesis between the spots.

The FMOC group is then removed (de-blocking) from the beta alanine and synthesis of the desired peptides begins. Beta alanine is used because it provides a uniform start to the synthesis and acts, along with the PEG, as a spacer. 40 nMoles of incoming FMOC protected residue are used to achieve coupling. Activation occurs via a DIC/HOBT strategy. De-blocking, washing, acetylation and final side-chain deprotection steps are performed manually. Three to four residues can be coupled in an average day. The membranes are labeled and grided and you are provided with a printout and file that details which sequences are contained on each spot.

At least 20 spots on one membrane must be dedicated to priming the amino acid deliveries - one spot for each amino acid that you use. In other words, if you are making a 1536 peptide synthesis run (four membranes) , the first 20 spots of the first membrane will be used for line priming if you are using only the 20 standard amino acids in your synthesis. Up to 44 amino acid reservoirs are available allowing for the synthesis of e.g. a membrane containing 20 L form residues, 20 D form residues plus four nonstandard residues such as PO3 Ser/Thr. We recommend that you also include an internal control such as an alanine scan of the HA epitope, which can be assayed later if necessary to resolve any quality issues.

References:

Tetrahedron, Vol. 48, No. 42, pp 9217-9232, 1992, Ronald Frank, "Spot synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support."

Tetrahedron Letters, Vol. 31, No. 40, pp 5811-5814, 1990, Andrew Bray, N. Joe Maeji and H. Mario Geysen, "The simultaneous multiple production of solution phase peptides; assessment of the Geysen Method of simultaneous peptide synthesis."

Some Uses for ABIMED Peptide Arrays

Antibody binding assays to determine specific epitopes
Mapping phosphorylation, sulfatation, glycosylation sites High resolution characterization of binding motifs - peptide to - (protein / DNA / metals) interactions Assays requiring free peptides. e. g. synthesizing peptide antigens for making antibodies Protease assays - peptides are stained with dye, then cut out and placed in separate wells of a microtitre plate. The release of the marker dye upon treatment with protease is a measure of the enzymatic activity
Amino Acid Replacement


Example Arrays

Quality Control

We recommend that you make short peptides (no longer than12 mers). The incorporation of phospho residues further than four to five amino acids from the N-terminal will severely degrade the sequence. Arginine residues are somewhat difficult to couple. Methione may oxidize. Understand that this apparatus is a very crude peptide synthesizer!

You can quality control the arrays by engineering a cleavable linker such as one of these taken from the Nova Biochemical catalog:

  • FMOC Rink Linker - C-terminal amide
  • FMOC amino photolinker - photocleavable amide
  • FMOC N methoxy 3 aminopropionic acid - (Weinreb Linker)

Example cleavable linker test peptide spot: APYDVPDYA - FMOC Rink Linker - beta alanine- PEG - cellulose

After cleavage, peptides can be analyzed by:

  • Mass spectrometry
  • HPLC
  • Protein sequencing
  • Amino acid analysis

An easier quality control is to engineer an epitope tag and probe with (commercially) available antibody. For example:

  • HA Tag: YPYDVPDYA influenza hemagglutinin epitope Monoclonal antibody HA.11
  • N-Myc Tag: SPYVESEDAPPQKC (aa 327-339) of human N-myc.

Example: alanine scan of the HA tag. Check with antibody:

APYDVPDYA
YAYDVPDYA
YPADVPDYA
YPYAVPDYA
YPYDAPDYA
YPYDVADYA
YPYDVPAYA
YPYDVPDAA
YPYDVPDYA

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