Custom Peptide Array Production Services

As an important component of our unique integrated suite of proteomics services, Kinexus offers custom peptide array synthesis to assist our clients in their discovery programs. Peptide arrays are powerful tools for the investigation of protein-protein and drug-protein interactions. Screening peptides for potentially active compounds with peptide arrays is a very convenient method for basic and applied research such as drug development. We describe here some of the principals and applications of peptide microarrays. A range of diverse peptide array-based services are available from Kinexus, and this package contains all of the information and forms required for our clients to utilize these services. For convenience, the various forms are available in fillable MS-Word versions that can be downloaded directly from the Kinexus website at http://www.kinexus.ca/ourServices/proteinAndPeptide/index.html or requested by e-mail from peptides@kinexus.ca.

For peptide-based drug design, with peptide microarrays it is possible to screen a high number of peptides on a small chip. However, due to the miniscule amounts of peptides synthesized directly on chips, and because of interactions of the peptides with the chip surface, this approach has proven to be difficult and often unreliable. These disadvantages can be overcome using peptide macroarrays on cellulose membranes. These are useful for solid-phase screening as well as solution-phase assays. Cellulose membranes are porous, hydrophilic, flexible and stable in organic as well as aqueous solvents. These properties make cellulose paper very useful for biochemical and biological studies in aqueous as well as in organic media and are a major reason why cellulose is still the most widely used material for macroarray membranes. Several hundred papers about applications using this method have been published. The spot densities of arrays range from a few up to approximately 8000 peptides.

Common cellulose membranes are not stable against harsh chemical conditions. Stable cellulose membranes are commercially available. Another development is the use of trifluoroacetic acid-soluble cellulose membranes. Peptides synthesized as macroarray on those membranes can be dissolved as peptide-cellulose conjugates and transferred in a large number of copies onto microarray glass slides keeping a three-dimensional structure with the high accessibility of cellulose-bound peptides (CelluSpots™).

The cellulose paper is usually functionalized with amino components like amino acids or amines. The attachment to cellulose is carried out either via ester or ether bonds. The ether bond is stable under common probing conditions, whereas the ester bond is labile and some loss can be expected during the probing. This is why ester-type are more useful for generating peptides cleaved from the membrane in order to use them in solution assays.

The synthesis of the peptides is carried out chemically, starting with the C-terminal amino acid. The monitoring the coupling process takes place by staining the spots with bromophenol blue after selected coupling cycles. The very large number of different peptide sequences that could be synthesized on a macroarray means that coupling rates and amount, as well as purity of the peptides may vary. The synthesis quality of peptides using this technique is comparable to other solid-phase peptides synthesis methods until a length of 15 amino acids. The synthesis of longer peptides can lead to significant lower quality and quantity.

In contrast to biological systems, the chemical synthesis of peptides facilitates the introduction of non-natural amino acids and other organic building blocks. Also the selected use of modified amino acids (e.g phosphoamino acids) is possible. Additionally, post-synthesis modifications like acetylation, cyclizations of fluorescence labeling can be performed.

After the final synthesis step the peptides are displayed as free peptides bound to the cellulose with their C-terminus. Depending on the screening purpose, there are different format of peptide sets (e.g. epitope mapping, substitution analysis etc.) The probing of such peptide macroarrays can be performed similar to other dot-blot techniques.

We offer membranes with ester-type modifications as well as membranes with the ether-type.

The ester-type membranes are usually modified with an amino acid. Our standard modification is either with beta-alanine or glycine. But we also can use other modifications on demand. This type of membranes is very useful for the production of free peptides adsorbed by the membrane in order to use them in solution assays. For that purpose we would synthesize peptides in large dots big enough to get punched out using a common hole puncher. After cleavage from the cellulose, the peptides at the C-terminus will retain the amino acid from the membrane modification. The peptides can be easily dissolved in appropriate solvent. The amount of each peptide is between 200 and 300 nmol for a standard modification.

We offer two types of ether-type modified membranes: N-CAPE and TOTD membranes. N-CAPE (N-modified cellulose aminopropyl ether) membranes have a short aminopropyl molecule between the cellulose and the peptide. TOTD (trioxatridecanediamine) membranes display three polyethylene-glycol units (PEG-3) between the cellulose and the peptides, which makes it more hydrophilic. This should result in less unspecific signals and background caused by hydrophobic interactions. The peptides are stable attached to either membranes and no loss on binding activity is expected.

Additionally we offer the use of chemically stable membranes, which we purchase from German suppliers. Those membrane are stable also against harsh chemical treatments, i.e. during strong regeneration procedures.

Depending on the purpose of the screening, different array formats can be generated. The standard size of the spots containing peptides is between 1 and 2 mm in diameter (small spots). For solution arrays, we synthesize the peptides in spot with a diameter of 5-7 mm (large spots). In this section, we present some array techniques to screen for active peptides.

Epitope Mapping (Peptide Scan) - CPAP1

Epitope mapping is a very useful method for screening a known protein sequence for biologically active regions (e.g. epitopes for antibody binding). The peptide sequences are generated by shifting a frame with a distinct peptide length over a protein sequence of interest. A peptide length between 10 and 15 amino acids is commonly used. Shifting of the frame between 1 and 3 amino acids is recommended; the smaller the shift the more precise will be localization of the binding region.

Substitution Analysis (Replacement Analysis, Mutational Analysis) - CPAP2

Substitution analyses are used for investigation of the importance of amino acids and their possible exchanges in a known peptide sequence. The sequence of peptides in this array will be generated by successive systematic substitution of each amino acid by other amino acids or building blocks of interest. Our standard substitution analysis will be performed by the systematic exchange of all positions in the known peptide sequence by all 20 common amino acids. On demand, we would also perform substitution analyses by using non-natural amino acids or other organic building blocks. Number of peptides on the array are dependent on the starting length of the parent (wild-type) peptide, and the number of amino acids/building blocks used for the substitution.

Truncation Analysis (Length scan) - CPAP3

To investigate the minimum possible length of an active peptide while maintaining activity, variations of the peptide sequence are synthesized by systematic shortening by one amino acid residue at each step from the C-terminus, N-terminus, and both termini simultaneously. Number of peptides on the array are dependent on the starting length of the parent (wild-type) peptide.

Random Peptide Library - CPAP4

To screen for active peptides without prior knowledge of a starting sequence, a random peptide library can be used. This array contains randomized peptide sequences. Compared with the combinatorial peptide library, the advantage of the random peptide array is that the peptides on each spot are unique, providing the possibility of higher individual activity. The disadvantage is in the relative low number of synthesized peptides available for screening. Our standard random peptide library contains 1200 peptide sequences with a length of 12 or 15 amino acids. But a synthesis of random peptide libraries based on other parameters is feasible.

Combinatorial Peptide Library - CPAP5

A very powerful method for screening active peptides without knowing the actual sequence is the combinatorial peptide library. Using combinatorial libraries, screening begins in theory with the entire pool of possible peptide sequences. Due to the impossibility of synthesizing all peptides as single sequences (for instance all possible 6-mers of the common 20 Lamino acids would result in 64,000,000 peptide sequences), in the first screening rounds a mixture of all possible amino acids and building blocks of interest would be used at most positions. To achieve distinct single sequences, a deconvolution over several synthesis and screening rounds is necessary. In practice, for the first round of synthesis and screening all but two positions include mixtures of amino acids or building blocks of interest. Following that, each peptide consists of a sequence with several mixed positions and two defined amino acids. The number of synthesized sequences is a combination of the number of used amino acid in these two defined positions. For instance, if at these two positions the common 20 L-amino acids were used, one would obtain 20x20=400 sequences. For a better overview the spots in an array would be arranged in a chessboard pattern with 20 rows and 20 columns. In each single row would represent all amino acids used at one position and combined it with the column-wise display for the other position. By screening one would obtain signals on spots with active sequence patterns. After the first synthesis and screening round there are two possibilities: First, one can combine different sequence patterns to a complete sequence. The second possibility is to select the sequence pattern with the strongest activity, keep the amino acid motif for these two positions, and perform a new synthesis and screening round for deconvolution of other two mixture positions. This repeating of deconvolution, synthesis and screening cycles proceeds until one obtains a complete sequence in which all amino acid positions are defined. The standard size of peptides in a combinatorial library is 6- to 8-mers, which would need 3 or 4 synthesis and screening rounds to achieve defined peptide sequences. The number of spots depends on the number of amino acids used for the combinations.

Cluster Peptide Library - CPAP6

To reduce the number of possibilities, combine several amino acids of similar properties in clusters (e.g. hydrophobicity – Ile, Leu, Val; or steric similarity – Ser, Cys, Abu). These clusterered amino acid peptide libraries provide easier and faster screening of large peptide libraries. It is a very useful alternative screening method to combinatorial and random peptide libraries. It is of course necessary to resolve these clusters at the end of the screening process.

Loop Scan (Cysteine Scan) - CPAP7

To stabilize loop structures or to increase their resistance to proteolytic digestion, it is convenient to cyclize peptides. There are two main types of cyclizations – cyclization via cysteines forming a disulfide bridge and cyclization via a pair of peptide amino and carboxy groups to form an amide bond. In both cases, a pair of amino acids are involved. If they are not present in the original peptide sequence, they must be inserted or two existing amino acids should be replaced which could lead to a loss of activity. Therefore, it is necessary to investigate the effect of the insertion/exchange and cyclization on the activity of the peptide. Our standard loop scan is a cysteine loop scan that contains a set of all possible combinations of insertions or replacements using a pair of cysteines. If requested, we could also perform an amide loop scan. Number of peptides on the array are dependent on the starting length of the parent (wild-type) peptide.

Customized Peptide Library - CPAP8

Our CPAP8 service offers also to synthesize a peptide macroarray on cellulose with a set of peptide sequences developed by our customers.

We also offer soluble peptides absorbed on the cellulose produced from each of the CPAP1 to CPAP8 services, but each peptide must be generated in large spots. The released peptides can be punched out and dissolved in an appropriate solvent and can be used for solution assays. Due to the modification of the membrane, these peptides are usually delivered as peptide amides with an additional amino acid C-terminal attached to the sequence. Standard amino acids for this additional amino acid are beta-Ala or Gly. The peptides attached to the cellulose can be delivered as a sheet or as a set of small paper discs.

We are pleased to offer our customers the possibility of using CelluSpots™ microarrays. This microarray consist of peptide-cellulose conjugates on each spot. It combines the advantages of a common microarray on glass slides with the high accessibility and three-dimensional structure of cellulose-bound peptides. The three-dimensional structure contains up to 100 times more peptide per area than conventional monolayer deposition.

Up to 384 spots could be printed in duplicates. An order for this type of arrays is useful, if many copies of arrays with many peptides are needed. Additionally, we offer ready-to-screen kinase-substrate peptide microarrys from annotated phosphorylation sites produced with the CelluSpots™ method.

The turnaround time for these services is estimated to be 3 to 4 weeks. However, this could varies slightly according to the size of the and the demand of the service at the time when the is placed. Each arrayed will be delivered with a comprehensive report.

CPAP1-8 Custom Peptide Arrays on Cellulose Membranes

Kinexus Custom Peptide Array Production (CPAP) Services offers different types of macroarrays. The price depends on the number of peptides and their length. The following prices in U.S. dollars are per amino acid residue and peptide for small spots (for larger spots we will charge 50% more):

CPAPA - CelluSpots™ Custom Peptide Arrays on Glass Slides

Minimum order volume is $ 300.00 (without membrane).

• Standard format 26x76 mm glass slides covered with white adhesive foil
  • Up to 384 unique spots printed in duplicates
  • 1.2 mm spot to spot distance
  • Contains control peptides and location marks

CPAPB - CelluSpots™ Kinase Substrate Peptide Arrays on Glass Slides

Ready to screen arrays with protein-tyrosine and protein-serine/threonine kinase substrates from annotated phosphorylation sites. This is an ideal tool to characterize substrate specificities of kinases, to compare kinases, to identify potential autophosphorylation sites, or to screen for kinase inhibitors.

All customers are required to complete the following forms for each order placed:

• Kinexus Proteomics Services Agreement.
• Service Order Form (CPAP-SOF). The Service Order Form (SOF) allows us to obtain client contact and billing information and establish the cost of the order.
• Service Identification Form (CPAP-SIF). The Service Identification Form (SIF) permits us to determine which specific Custom Peptide Array Production
• Services are requested.

A. Kinexus Proteomics Services Agreement

• A signed Kinexus Proteomics Services Agreement is required before your first order with Kinexus can be processed.
• This Agreement is required to be signed and dated by an authorized representative, typically a Senior Officer, Senior Scientist, or Principal Investigator, before the first order can be processed, but does not have to be signed again for repeat orders. The Kinexus Service Agreement is typically valid for 15 years. If you require changes or modifications to be made to our standard Service Agreement, please email us at sales@kinexus.ca to request a Microsoft Word version of the document so your requested changes can be made directly into the agreement and emailed to us for our final approval.

B. Service Order Form (CPAP-SOF)

Please ensure:

• Shipping address and contact name and numbers are specified.
• Billing information is completed.
• Any promotional vouchers or quotations are listed in the billing sections.
• Include a Purchase Order, Visa or MasterCard number for payment.
• The form is signed and dated.

C. Service Identification Forms (CPAP-SIF)

Note that:
Fillable MS-Word versions of all of these CPAP-SIF forms and the CPAP-SOF are available for download from the Kinexus website at the bottom of the webpage http://www.kinexus.ca/ourServices/proteinAndPeptide/index.html. Such electronically completed forms can be sent via email for rapid processing of orders. For direct request of such fillable MS-Word forms as well as for all enquiries related to peptide synthesis and array technical/research issues, work orders, and service fees, please contact Dr. Dirk Winkler by email at peptides@kinexus.ca or by phone at 604-323-2547 Ext.17.

• The service identification forms (SIF) allows us to track all of the various services to be used within an order. For each of the different array types there is a separate order form:

• For each CPAP service used, please assign a unique name (Client ID Name) to be entered on the Service Order Form (CPAP-SOF).

When Kinexus receives the appropriate completed forms described above, We will review the supplied information to make sure that the forms are completed correctly. You will receive a confirmation of the specifics for your order, including pricing. We will not proceed with your order until we have received verification of your approval to go ahead and process your order.

Kinexus offers also a testing service for synthesized macroarrays on cellulose. We would perform this testing along with a negative control. The necessary protein/antibody is either provided by the customer or, if available, we would purchase it, with additional cost to the client. Unless there are other instructions from the customer, the treatment will be carried out similar to other blotting techniques. The bound protein would be detected via HRP/chemiluminescence, but staining would also be possible.

The price would be at US $ 500 per sample (excluding possible charge for purchase of protein). The probing would be carried out only if we receive payment of 50% of the total price in advance after the successful synthesis.

Please contact Dr. Dirk Winkler by email at peptides@kinexus.ca or by phone at 604-323-2547 Ext.17.