Services

Peptide Synthesis

Large Scale (Individual) (0.1 - 0.25mmol)

General Information

We are using an ABI Model 433A peptide synthesizer with on-line UV monitoring of FMOC deprotection and conditional cycle feedback. This instrument offers two scales of synthesis: 0.1 and 0.25 mmol. (We may reduce the scale somewhat for peptides with long and/or difficult sequences.) Starting with polystyrene resin, the peptide is built "backwards," from the C-terminus to the N-terminus. After synthesis, the peptide is cleaved from the resin, yielding the crude form of the peptide. Generally, this type of synthesis yields crude peptides with 85% purity, but HPLC purification can be done if higher purity is desired. Average peptide lengths are 10-20 amino acids.

Certain combinations of amino acids cause aggregation and result in low coupling yields and poor quality peptides. Usually these sequences occur in the 7 to 15 amino acid stretch emanating from the C-terminal. Some difficult sequences are obvious to people who have peptide synthesis experience, but most are not. Because the rate at which FMOC deprotection occurs is proportional to the rate at which the incoming residue will couple, UV on-line monitoring is crucial for the synthesis of long peptides. The beauty of our synthesizer is that it can determine when aggregation occurs and modify reaction vessel conditions to compensate by extending the deprotection and coupling periods. This results in high quality short and long peptides.

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N- & C-terminal Chemistry

Standard N-terminal chemistry is either free amine or acetylation, and standard C-terminal chemistry is either free acid or amidation. You may want to calculate the isoelectric point (pI) of the sequence and consider the pH of the buffer that you will be working in prior to choosing terminal chemistry. By blocking the terminals, you may end up with a peptide that is not very soluble in your working conditions. However, you may want to neutralize the terminal(s) to mimic the appearance of the peptide in vivo for better antibody recognition. See the Antibody Production Notes and References section.

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Modified Residues

Modified residues, such as unusual amino acids, can be incorporated into a synthesis. If we do not have a residue in the lab, we will order it, and the user will be charged for the price of the order.

Commonly-used modified residues:

- Phosporylated Serine, Threonine, and Tyrosine (pS, pT, pY)
- Aminohexanoic acid (Ahx)
- Lysine-Dde (K-Dde)
- Beta Alanine (B-Ala)
- D-form amino acids

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Dye Labeling

We can attach dyes to peptides, either at the N-terminus or at a Lysine residue. The most common dyes used are fluorescein (absorbs @490nm) and TAMRA (absorbs @540nm). This procedure is done post-synthesis, but while the peptide is still attached to the resin. Cleavage will then follow.

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Biotinylation

Biotin can be added to the N-terminus of a peptide or at a Lysine residue. This procedure is done post-synthesis, but while the peptide is still attached to the resin. Cleavage will then follow.

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Desalting

The peptide fraction of the crude material consists of the ideal peptide, traces of deletion peptides and modified peptides created during side chain deprotection and cleavage from the starting resin. Since purification is time consuming and not always necessary, we recommend that you experiment with the crude or desalted product before requesting HPLC purification. Most researchers use the crude peptide for coupling to carrier protein for immunization. We do not do desalting in the laboratory, but it can be done by gel filtration chromatography (e.g. G10 or G25 columns). Disposable gel columns are available from companies like BioRad and Pharmacia/Amersham. For larger scale desalting the packing material can be purchased separately and you can pour your own column. Desalting protocols are available from manufacturers catalogues and from peptide synthesis reference books such as Stewart and Young's "Solid Phase Peptide Synthesis."

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HPLC Purification

If high purity peptide is required, we offer HPLC purification of peptides. Our HPLC purification consists of taking approximately 100 mg of crude peptide (the amount depends on the complexity of the analytical HPLC trace of the crude product) and injecting that material onto a preparative scale reverse phase column. We collect all column fractions and pool those containing the ideal product. The amount of pure material recovered depends on the sequence but usually is better than 50% of that loaded. The pure peptide is provided along with any crude peptide remaining from the purification process.

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Antibody Production Notes & References

See Antibody Production Notes & References Page.

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Quality Control

We provide you with an HPLC trace of the peptide to assess purity by UV along with mass spectrometer spectra. HPLC and MS are performed under slow gradient / high resolution conditions. If you have a peptide from an outside source and are suspicious about quality we will be happy to subject an aliquot to our QC procedure.

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Cost

See Pricing Chart.

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How to Order

See Ordering Information.

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The Final Product

The peptide, in both crude and purified forms, is given out as dry powder with a specified weight. HPLC and mass spectrometry data are provided as well.

To determine how much peptide is present: 

1. Assume that about 75% by weight is peptide, with the remainder being H20 and TFA salt.

2. If there is Tyr, Trp, Phe, or Cys in the sequence, you can use the extinction coefficients for those amino acids to calculate the amount present.  (@ 280nm, eY=1280, eW=5690, eC=120, eF=2 e=extinction coefficient in units of Lmol-1cm-1)

epeptide=aeY + beW + ceC + deF, where a=# Y in sequence, b=#W, c=#C, d=#F
Use c=A/(epeptide*l), where c=molar concentration of peptide in mol/L, A=absorbance at 280nm, and l=path length of cuvette in cm

3. Amino acid analysis is the most accurate method. We can help you interpret the AAA data if you like. We suggest these analysis labs:

AAA Laboratory  (Lowell H. Ericsson) 6206  89th Ave. SE Mercer Island, WA  98040-4599 206 364-3446 fax 206 236-6305	Jay Gambee AAA Service Laboratory, Inc. 14865 SE Regner Terrace Dr. Boring, OR 97009-1258 Ph: 503 658-6997 Fax: 503 558-9297 Email: aaaservs@teleport.com
AAI 1206 North 23rd Street Wilmington, NC  28405 (910) 251-6700 or (800) 575-4AAI	Dr. Mary K. Young Director Mass Spectrometry and Protein Microsequencing Core Facility BRI / City of Hope National Medical Center Duarte, CA 626-256-4673 X62601

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Small Scale (Multiple) (1-10umol)

General Information

We use the Intavis Model MultiPep multiple peptide synthesizer to make up to 192 peptides simultaneously. The synthesis scale is 1-10umol, with 5umol being the most common. This service is ideal for peptide library and screening experiments. Applications include protein binding interactions, epitope mapping, enzyme inhibition and immunodiagnostics. Starting with tentagel resin, the peptide is built "backwards," from the C-terminus to the N-terminus. After synthesis, the peptide is cleaved from the resin, yielding the crude form of the peptide. Generally, this type of synthesis yields crude peptides with 80-85% purity, but HPLC purification can be done if higher purity is desired. Average peptide lengths are 10-20 amino acids. In a typical run, 0-5% of the peptides fail to synthesize properly due to difficulties in coupling caused by the specific amino acid sequence of the peptide.

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N- & C-terminal Chemistry

Standard N-terminal chemistry is either free amine or acetylation, and standard C-terminal chemistry is either free acid or amidation. You may want to calculate the isoelectric point (pI) of the sequence and consider the pH of the buffer that you will be working in prior to choosing terminal chemistry. By blocking the terminals, you may end up with a peptide that is not very soluble in your working conditions. However, you may want to neutralize the terminal(s) to mimic the appearance of the peptide in vivo for better antibody recognition. See the Antibody Production Notes and References section.

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Modified Residues

Modified residues, such as unusual amino acids, can be incorporated into a synthesis. If we do not have a residue in the lab, we will order it, and the user will be charged for the price of the order.

Commonly-used modified residues:

- Phosporylated Serine, Threonine, and Tyrosine (pS, pT, pY)
- Aminohexanoic acid (Ahx)
- Lysine-Dde (K-Dde)
- Beta Alanine (B-Ala)
- D-form amino acids

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Dye Labeling

We can attach dyes to peptides, either at the N-terminus or at a Lysine residue. The most common dyes used are fluorescein (absorbs @490nm) and TAMRA (absorbs @540nm). This procedure is done post-synthesis, but while the peptide is still attached to the resin. Cleavage will then follow.

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Biotinylation

Biotin can be added to the N-terminus of a peptide or at a Lysine residue. For small scale peptides, biotinylation is done on the peptide synthesizer.

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Desalting

The peptide fraction of the crude material consists of the ideal peptide, traces of deletion peptides and modified peptides created during side chain deprotection and cleavage from the starting resin. Since purification is time consuming and not always necessary, we recommend that you experiment with the crude or desalted product before requesting HPLC purification. Most researchers use the crude peptide for coupling to carrier protein for immunization. We do not do desalting in the laboratory, but it can be done by gel filtration chromatography (e.g. G10 or G25 columns). Disposable gel columns are available from companies like BioRad and Pharmacia/Amersham. For larger scale desalting the packing material can be purchased separately and you can pour your own column. Desalting protocols are available from manufacturers catalogues and from peptide synthesis reference books such as Stewart and Young's "Solid Phase Peptide Synthesis."

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HPLC Purification

If high purity peptide is required, we offer HPLC purification of peptides. For small scale peptides, our HPLC purification consists of taking 5umol of peptide and injecting that material onto a preparative scale reverse phase column. We collect all column fractions and pool those containing the ideal product. The amount of pure material recovered depends on the sequence but usually is better than 50% of that loaded.

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Antibody Production Notes & References

See Antibody Production Notes & References Page.

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Quality Control

We provide you with mass spectrometry data and, upon request, HPLC data. Usually with the small scale peptides, the mass spectrometry data is a sufficient quality control. MS and HPLC are performed under high resolution/slow gradient conditions.

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Cost

See Pricing Chart.

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How to Order

See Ordering Information.

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The Final Product

The peptide, in both crude and purified forms, is given out as dry powder . Mass spectrometry data is provided as well (HPLC data is provided upon request).

To determine how much peptide is present: 

1. Assume that 50% of the synthesis scale is present for each peptide.

2. Assume that about 75% by weight is peptide, with the remainder being H20 and TFA salt.

3. If there is Tyr, Trp, Phe, or Cys in the sequence, you can use the extinction coefficients for those amino acids to calculate the amount present.  (@ 280nm, eY=1280, eW=5690, eC=120, eF=2 e=extinction coefficient in units of Lmol-1cm-1)

epeptide=aeY + beW + ceC + deF, where a=# Y in sequence, b=#W, c=#C, d=#F
Use c=A/(epeptide*l), where c=molar concentration of peptide in mol/L, A=absorbance at 280nm, and l=path length of cuvette in cm

4. Amino acid analysis is the most accurate method. We can help you interpret the AAA data if you like. We suggest these analysis labs:

AAA Laboratory  (Lowell H. Ericsson) 6206  89th Ave. SE Mercer Island, WA  98040-4599 206 364-3446 fax 206 236-6305	Jay Gambee AAA Service Laboratory, Inc. 14865 SE Regner Terrace Dr. Boring, OR 97009-1258 Ph: 503 658-6997 Fax: 503 558-9297 Email: aaaservs@teleport.com
AAI 1206 North 23rd Street Wilmington, NC  28405 (910) 251-6700 or (800) 575-4AAI	Dr. Mary K. Young Director Mass Spectrometry and Protein Microsequencing Core Facility BRI / City of Hope National Medical Center Duarte, CA 626-256-4673 X62601

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Cellulose Peptide Array (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."

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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

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Antibody Production Notes & References

See Antibody Production Notes & References Page.

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Example Arrays

Some Sample Array Sequences

Frame shift peptides from protein - The arrayer can be used to generate comprehensive sets of peptides from imported protein sequences. The seq.12.2 command tells the instrument to make 12mer peptides from the protein sequence pasted in below, with a frame shift of two residues. The C-terminal peptide is generated regardless of frame shift

.seq,12,2

P I S P I E T V P V K L K P G M D G P K V K Q W P L T E E K I K A L V E I C T E M E K E G K I S K I G P E N P Y N T P V F A I K K K D S T K W R K L V D F R E L N K R T Q D F W E V Q L G I P H P A G L K K K K S V T V L D V G D A Y F S V P L D E D F R K Y T A F T I P S I N N E T P G I R Y Q Y N V L P Q G W K G S P A I F Q S S M T K I L E P F R K Q N P D I V I Y Q Y M D D L Y V G S D L E I G Q H R T A D Q

.end

Below are listed the first few peptides from this run.

97 P I S P I E T V P V K L 1293.9
98 S P I E T V P V K L K P 1308.9
99 I E T V P V K L K P G M 1312.2
100 T V P V K L K P G M D G 1242.1
101 P V K L K P G M D G P K 1267.2
102 K L K P G M D G P K V K 1298.2
103 K P G M D G P K V K Q W 1371.3
104 G M D G P K V K Q W P L 1356.3

Generation of different sized peptides - The command below instructs the instrument to make all 8mers through 4mers from the entered peptide sequence with a frame shift of one residue.

".seq,n1,n2,n3" and ".end", where n1 defines the longest peptide

seq,8,1,4H I D D E N E P I T O P E S E Q E N C E.end

231 O P E S E Q 692.5

Replacement set or Analog Peptides - The command below instructs the arrayer to substitute any desired set of amino acids positioned between the asterisks in "analog,* *" at the position designated by the asterisk in the peptide sequence.

.analog,*= replace star with each of the residues defined in the list.

.analog,*=*

E * A M P L E

275 E A A M P L E 760.1
276 E R A M P L E 845.2
277 E N A M P L E 803.2
278 E D A M P L E 804.1
279 E C A M P L E 792.2
280 E Q A M P L E 817.2
281 E E A M P L E 818.1
282 E G A M P L E 746.1
283 E H A M P L E 826.2
284 E I A M P L E 802.2
285 E L A M P L E 802.2
286 E K A M P L E 817.2
287 E M A M P L E 820.3
288 E F A M P L E 836.2
289 E P A M P L E 786.2
290 E S A M P L E 776.9
291 E T A M P L E 790.1
292 E W A M P L E 875.3
293 E Y A M P L E 852.2
294 E V A M P L E 788.2

Library Peptides

.analog,*=A,B,C

L I B R A R Y * *

295 L I B R A R Y A A 976.3
296 L I B R A R Y B B 920.1
297 L I B R A R Y C C 1040.5

Individual Peptides

.pep

T E S T P E P T I D E

E X A M P L E P E P T I D E

298 T E S T P E P T I D E 1219.2
299 E X A M P L E P E P T I D E 1584.2

You can browse the ABIMED manual in our lab for a complete list of options.

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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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Cost

See Pricing Chart.

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How to Order

See Ordering Information.

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