PROTEIN ID BY MASS SPECTROMETRY CUSTOMER INFORMATION PACKAGE

Transcription

1 MASS SPECTROMETRY SERVICES PIMS Version 10NO11 PROTEIN ID BY MASS SPECTROMETRY CUSTOMER INFORMATION PACKAGE For identification of antibody cross-reactive proteins detected by immunoblotting. Toll free: KINEXUS or Facsimile:

2 TABLE OF CONTENTS Protein Identification by Mass Spectrometry Services Description PDF Page No. 1. Introduction... 3 Sample Preparation 2. Tissue Preparation Cell and Tissue Lysates Preparation of Cultured Cell Lysates...9 A. Adherent Cell Lysates...9 B. Suspension Cell Lysates Preparation of Cell Pellets...9 A. Adherent Cells...9 B. Suspended Cells Shipping and Pricing 6. Preparation for Storage and Shipping of Samples Shipping Information Pricing Information Follow Up Services and Forms 9. Follow Up Services Forms to be Complete Forms to Complete and Return with your Samples 11. Service Order Form (PIMS-SOF) Service Identification Form (PIMS-SIF) Client-Supplied Non-Confidential Sample Description Form - Lysates (NSDF-LY) Client-Supplied Confidential Sample Description Form - Lysates (CSDF-LY) Client-Supplied Non-Confidential Sample Description Form - Antibodies (NSDF-AB) Client-Supplied Confidential Sample Description Form - Antibodies (CSDF-AB) Commercial Invoice (required for customers outside of Canada) Kinexus Service Agreement (first time customers only)...23

3 KINEXUS PROTEIN IDENTIFICATION BY MASS SPECTROMETRY SERVICE 1. INTRODUCTION General Methodology Protein identification by mass spectrometry (PIMS) has been made feasible due to the convergence of two recent technological advances. The first is the improvement in sensitivity and resolution of modern mass spectrometers, allowing accurate mass measurements of peptides in femtomolar to attomolar concentrations at resolutions better than 0.1 Da in 1000 Da. The second, is the advent of high throughput DNA sequencing leading to whole genome sequence data for several species such as mouse, rat, and human. By obtaining accurate masses of peptides and matching these to predicted gene product masses, the identity of an unknown protein can often be determined Our PIMS Service is positioned relative to the other services offered by Kinexus as a follow on supplement to our core Kinex antibody microarray (KAM) and Kinetworks multi-immunoblotting services, and is a means of obtaining additional information from these early stage protein profiling services. These initial stage services utilize Kinexus unique antibody microarrays and multi-immunoblots to track the differential binding of proteins in lysates prepared from cells and tissues. The results can provide some valuable antibody information relating to differences in protein expression, phosphorylation and protein-protein interactions, but due to the ability of antibodies to crossreact with non-target proteins, this information is not always sufficient to positively link an antibody to the protein. Our PIMS service can establish a positive link, and allow follow-up studies to validate an interesting protein biomarker in larger numbers of biological specimens. Protein Identification by Mass Spectrometry is carried out by immunoprecipitation of the protein of interest using an antibody identified in the KAM or subsequent Kinetworks multi-immunoblotting services. Then, the immunoprecipitate is subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to resolve a novel-sized protein, meaning a protein different in size from the expected antibody target. The protein band of interest is then excised from the SDS-PAGE gel, subjected to trypsin digestion to produce a characteristic set of tryptic peptide fragments, and then analyzed by high-resolution mass spectrometry to determine their accurate masses. A search of the appropriate protein sequence databases using a Mascot search ( for a matching mass pattern can then lead to a definitive identification of the protein. The mass spectrometry is performed on an LC-MS/MS using a Thermo Electron LTQ-Orbitrap. The methodology of the protein identification process is described in the following two figures. Figure 1 illustrates an experimental design using untreated versus treated cells or tissues. The untreated arm is an important control for identifying protein bands that respond to treatment (and may be potential biomarkers). Figure 2 illustrates an alternative design where untreated control cells or tissues may not be available in sufficient quantities. In this case, the agarose beads without conjugated antibody are used as a negative control for immunoprecipitation. This allows the differentiation between protein bands that bind nonspecifically to the beads, versus proteins that bind specifically to the antibody. 10NO11 Protein Identification by Mass Spectrometry Customer Information Package 3 Kinexus Bioinformatics Corporation

4 Ab selected from Kinex Ab Microarray & Kinetworks Multi-immuno-blotting services showing a treatment response Conjugate Ab to agarose beads use for both treated and untreated arms of experiment Treated tissue or cell lysate Untreated tissue or cell lysate Excise Coomassie bluestained band present or stronger in treated versus untreated sample Immunoprecipitate with Ab plus beads SDS-PAGE Immunoprecipitate with Ab plus beads SDS-PAGE In-gel trypsin digestion of excised band Nano-HPLC to separate the multiple tryptic peptide fragments High resolution and sensitivity MS/MS to determine accurate masses of parent and Collision Induced Dissociation daughter peptides Accurate mass data used in MASCOT search of predicted parent-daughter mass combinations for tryptic protein fragments from selected species to give protein identification Figure 1. Methodology of protein identification by mass spectrometry using untreated cells in the negative control arm. 10NO11 Protein Identification by Mass Spectrometry Customer Information Package 4 Kinexus Bioinformatics Corporation

5 Tissue or cell lysate from treated arm of experiment Select Ab showing a treatment response from Kinetworks multi-immunoblots No Ab selected for negative control Conjugate Ab to agarose beads to give Ab plus Agarose beads alone give Ab minus beads Excise Coomassieblue stained band present in Ab plus but not in Ab minus Immunoprecipitate treated lysates with Ab plus beads SDS PAGE Immunoprecipitate treated lysates with Ab minus beads SDS PAGE In-gel trypsin digestion of excised gel band Nano-HPLC to separate the multiple tryptic peptide fragments High resolution and high-sensitivity MS/MS to determine accurate masses of parent and Collision Induced Dissociation daughter peptides Accurate mass data used in MASCOT search of predicted parent-daughter mass combinations for tryptic protein fragments of selected species to give protein identification Figure 2. Methodology of protein identification by mass spectrometry using agarose beads without antibody as a negative control. 10NO11 Protein Identification by Mass Spectrometry Customer Information Package 5 Kinexus Bioinformatics Corporation

6 Example Study Effects of a Compound on Protein Expression in Rat Lung A company studying the effects of a compound on lung function carried out a study where laboratory rats were subjected to the agent and then the lung tissue was analyzed using the Kinex TM antibody microarray service. Several antibodies were identified to recognize proteins that were elevated in the agent treated group versus the untreated control group. Four of these antibodies were selected to be subjected to our PIMS service.. Each antibody was conjugated to agarose beads and incubated with 4 mg of rat lung lysate protein from the compound-treated animals to immunoprecipitate the protein(s) that were potential biomarkers. The control used in this test was the agarose beads without antibody. Out of the 4 antibodies used for immunoprecipitation, there were two that revealed one or more gel bands in the antibody plus immunoprecipitate versus the antibody minus samples. The differentially affected bands were excised and subjected to trysin digestion and LC-MS/MS. One of these could be identified with high confidence in a Mascot search. The above example study illustrates some caveats with respect to the performance and success rate of the Kinexus PIMS Service. Firstly, an antibody identified in the Kinex TM antibody microarray, even if it is confirmed in the follow-up Kinetworks multi-immunoblots will not necessarily succeed in immunoprecipitating a differentially expressed protein band visible on a Coomassie blue-stained gel. The antibody microarray or immunoblot methods are much more sensitive than a Coomassie blue-stained gel, so it is possible that the immunoprecipitated protein is present below the detection limit of Coomassie blue. Secondly, when a Coomassie blue-stained protein band is visible as a differentially expressed protein, the identification by mass spectrometry does not always yield a high probability identification. Peptide masses be influenced, for example, by phosphorylation of the protein or a variety of other covalent modifications. Factors such as small tryptic peptide size and poor reverse-phase LC resolution will affect the degree of certainty for a given mass spectrometry protein identification. However, knowledge of the apparent molecular mass of the protein improves the likelihood of a correct identification. A large body of information and instruction is provided with this PIMS Services Customer Information Package. Your careful review of this package will ensure that we can offer the highest level of quality in providing our unique proteomics services to you. We have requested a lot of information from you regarding the preparation of your cell/tissue lysate samples so that we may share with other scientists in the future. For these rights, we have discounted our standard charges by ~40% with our Non-Confidential Pricing option. You should find that the proper entry of information into the various forms provided by Kinexus will also be useful for your own reference at a later date when you receive your PIMS results. Should you have any questions or concerns, we would be pleased to hear from you. Thank you in advance for letting Kinexus become one of your trusted research partners. 10NO11 Protein Identification by Mass Spectrometry Customer Information Package 6 Kinexus Bioinformatics Corporation

7 SAMPLE PREPARATION 2. TISSUE PREPARATION Lysis Procedure 1. Keep tissue on ice at all times. Rinse whole uncut samples with ice cold PBS to remove surface blood, and then cut into pieces less than 1 mm in size. 2. Carry out lysis procedure in batches using 0.5 ml of lysis buffer for approximately 125 mg wet weight of chopped tissue contained in a 2 ml microfuge tube. 3. Homogenize tissue in lysis buffer on ice for 6 intervals of 10 seconds each, pausing 10 seconds between, using an ultrasonic probe (Vibracell, Sonics & Materials Inc. Danbury CT, USA, 40 Watt output generator or equivalent) with a 1.3 cm horn and 3 mm tip, at a power output setting of about 10 Watts 4. Centrifuge homogenate at 90,000 to 110,000 x g for 30 min at 4 C in a Sorvall M120 Micro-Ultracentrifuge (55,000 rpm using rotor S120-AT2 or equivalent). 5. Transfer the resulting supernatant fraction to a new tube and carry out a protein assay. Using a commercial Bradford assay (available from Bio-Rad, catalogue number ) or using the standard protocol of Bradford (Bradford, M.M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal. Biochem. 72: ). Bovine serum albumin should be used as the protein standard. 3. CELL AND TISSUE LYSATES For the Protein Identification by Mass Spectrometry (PIMS) the preferred amount of lysate protein is 5 to 10 mg per sample submitted for immunoprecipitation at a concentration of 10 to 50 mg/ml. If this is not possible, then smaller amounts can be handled using modified procedures, but with lower probability of success. The samples must be frozen and shipped to Kinexus on dry ice fresh after protein quantification WITHOUT ANY SDS-PAGE SAMPLE BUFFER as the proteins are to remain in their native structure and nondenatured. Important general points to remember are that the cells or tissues should be processed quickly at 4 C or less. Homogenization should not be performed in too large a volume to obtain lysates at the concentration required. The detergent-soluble fraction should be obtained as quickly as possible after the cells or tissues are homogenized. Sonication is required and cannot be omitted. The highest centrifugal forces available should be used to generate the detergent-soluble fraction. The supernatants should be frozen as quickly as possible if a protein assay cannot be performed immediately. Standard Lysis Buffer mm MOPS, ph 7.0 adjusted using either 1 M HCl or 1 M NaOH (other ph buffering agents and be used as long as they do not contain reactive amine groups; TRIS cannot be used.); 2. 2 mm EGTA, ph 8.0 (to bind calcium); 3. 5 mm EDTA, ph 8.0 (to bind magnesium and manganese); mm sodium fluoride (to inhibit protein-serine phosphatases); mm beta-glycerophosphate, ph 7.2 (to inhibit protein-serine phosphatases); 10NO11 Protein Identification by Mass Spectrometry Customer Information Package 7 Kinexus Bioinformatics Corporation

8 6. 20 mm sodium pyrophosphate (to inhibit protein-serine phosphatases); 7. 1 mm sodium orthovanadate (to inhibit protein-tyrosine phosphatases); 8. 1% Triton X-100 (nonionic detergent to disrupt cellular membranes; 1% Triton X-100 can be substituted with 1% Nonidet P-40; Note: Do not add detergent if you intend to first prepare a subcellular fraction) mm phenylmethylsulfonylfluoride (to inhibit proteases); mm benzamidine (to inhibit proteases); µm pepstatin A (to inhibit proteases); µm leupeptin (to inhibit proteases); mm dithiothreitol (reducing agent to disrupt disulfate bonds, this concentration can be modified, depending upon objectives of experiment). Note 1: The protease inhibitors phenylmethylsulfonylfluoride, benzamidine, pepstatin A leupeptin and the reducing agent dithiothreitol must be added to lysis buffer immediately before use. For convenience, one may choose to use the Roche Complete Protease Inhibitor Cocktail Tablet, Mini catalog# in place of individual protease inhibitors. The lysis buffer must be used ice-cold. Note 2: Other lysis buffers commonly used for protein lysate preparation with non-ionic detergents should still be compatible with the service. Please contact a Kinexus Technical Sales Representative for more information on the appropriate types of lysis buffers to use for the PIMS Service or to request to have an aliquot of our lysis buffer for free if you can provide a courier account number to charge for the shipping costs. Our lysis buffer contains components 1-8, including phosphatase inhibitors (components 4-7) and 1% Triton X-100 but no protease inhibitors or dithiothreitol. Subcellular Fractions Depending upon required cellular fraction, it may be required to modify the lysis buffer. Total cellular fractionation: For quantitation of total cellular levels of cell signaling proteins, lysis and homogenization should be performed in the presence of a non-ionic detergent. We recommend the use of 1% Triton X-100 or 1% Nonidet P40, but comparable detergents are acceptable. Subcellular fractionation: Detergents should be omitted from the homogenization buffer if the subcellular distribution of cell signalling proteins is to be examined. If a particulate-solubilized fraction is to be analyzed, a microsomal pellet should be obtained following the initial homogenization and ultracentrifugation in the absence of detergent and subsequent removal of the cytosolic supernatant. In this instance, the cytosolic extract should be removed and the microsomal pellet should then be resuspended in the homogenization buffer containing 1% Triton X-100 or 1% Nonidet P-40 and subjected to homogenization and ultracentrifugation once again. The resulting detergent-solubilized microsomal fraction should be removed and immediately assayed for its protein concentration. 10NO11 Protein Identification by Mass Spectrometry Customer Information Package 8 Kinexus Bioinformatics Corporation

9 4. PREPARATION OF CULTURED CELL LYSATES A. Adherent Cell Lysates 1. From culture dishes containing a total minimum of cells; remove the medium by aspiration. 2. Rinse the cells twice with ice-cold PBS to remove serum proteins from the cells after the last rinse, remove as much PBS as possible by aspiration. For sensitive cells, the rinses can be done medium without serum proteins. 3. Add 200 µl ice-cold lysis buffer to 150 mm culture dish per sample (more lysis buffer can be added if cells are concentrated); (add 100 µl ice-cold lysis buffer to 100 mm culture dish) 4. Scrape the cells in lysis buffer, collect the cell suspension from the dishes and transfer it into a 1.5-mL microcentrifuge tube; 5. Sonicate on ice to rupture the cells and to shear nuclear DNA; A typical sonication would be for 6 intervals of 10 seconds each, pausing 10 seconds between, using an ultrasonic probe with a 1/8 th inch or 3 mm tip and power setting equivalent to about 10 Watts output (Vibracell VC40, Sonics & Materials Inc. Danbury CT, USA, 20 KHz 40 Watt maximum output generator or equivalent). This step is crucial and cannot be omitted; 6. Centrifuge the homogenate at 90,000 to 110,000 x g for 30 min at 4 C in a Sorvall M120 Micro- Ultracentrifuge (55,000 rpm using rotor S120-AT2 or equivalent); 7. Transfer the resulting supernatant fraction to a 1.5-mL microcentrifuge tube; 8. Assay sample for protein concentration using a commercial Bradford assay reagent (available from Bio- Rad, catalogue number ) or using the standard protocol of Bradford (Bradford, M.M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: ). Bovine serum albumin should be used as the protein standard. B. Suspended Cell Lysates 1. Place medium containing cells in appropriate sized tube and centrifuge at 500 x g for 2 minutes at 4 C in a swinging bucket benchtop centrifuge. Remove as much medium from the cell pellet as possible without disrupting cells; 2. Wash the pellet by gently resuspending the cells in ice-cold PBS, followed by centrifugation as above. Repeat once to ensure complete removal of serum; 3. Remove as much PBS as possible after the last wash; 4. Add an adequate amount of ice-cold lysis buffer to the sample (more lysis buffer can be added if the number of cells is high); 5. Follow the procedure for adherent cells from step PREPARATION OF CELL PELLETS An additional charge of $200 per sample will apply for submission of cell pellets to be processed at Kinexus. Please submit a sufficient number of cells ( > cells) for processing. A. Adherent Cells 1. Remove the medium and rinse the cells in dish with ice-cold PBS once; 10NO11 Protein Identification by Mass Spectrometry Customer Information Package 9 Kinexus Bioinformatics Corporation

10 2. Detach cells with trypsin as one does in passaging cells, followed by the addition of equal volume of medium; 3. Collect cells in a 15-mL conical tube and centrifuge at 500 x g for 2 minutes at 4 C in a swinging bucket benchtop centrifuge; 4. Wash the pellet twice with ice-cold PBS thoroughly, (the presence of serum from medium could skew the protein assay) and remove as much PBS as possible (the presence of liquid residue dilutes the sample and may also result in the damage of cells during freezing process); 5. Freeze the pellet for shipping. Pellet must be shipped on dry ice. B. Suspended Cells Simply follow steps 3-5 in Section 4A for for adherent cells and freeze the cell pellet immediately. Pellet must be shipped on dry ice. SHIPPING AND PRICING 6. PREPARATION FOR STORAGE AND SHIPPING OF SAMPLES For the Protein Identification by Mass Spectrometry (PIMS) the preferred amount of protein is 4 to 10 mg per sample submitted for immunoprecipitation at a concentration of 10 to 50 mg/ml. If this is not possible, then smaller amounts can be handled using modified procedures, but with lower probability of success. Please record the actual concentration and volume of each sample on the Sample Description Form (NSDF-LY or CSDF-LY). Samples should be stored in screw cap vials. The vials should be clearly labeled with an indelible marker with a unique identification number (recorded in the Sample Description Form), parafilmed, and then put into another support structure such as a 50-mL conical or centrifuge tube to provide extra protection during shipping. All samples must be shipped on dry ice. Approximately 5% of the time, it has been necessary for clients to re-send samples to Kinexus due to thawed samples at the time of arrival. This is most often due to insufficient dry ice for shipping and/or inadequate completion of shipping information. 7. SHIPPING INFORMATION The aforementioned procedure has been designed to reduce the use of shipping materials and courier costs, and to ensure that your precious samples arrive in a safe and stable form at our laboratory facilities. Note that clients are responsible for payment of courier costs. The sample vials should be sent to the address listed below by any express courier that accepts dry ice shipments. We recommend Federal Express for shipments originating in North America, and World Express is the preferred courier choice outside of North America. Ship the samples to the following address: Protein Identification by Mass Spectrometry Services Kinexus Bioinformatics Corporation Suite 1, 8755 Ash Street Vancouver, B.C. Canada V6P 6T3 Telephone: (604) Facsimile: (604) info@kinexus.ca 10NO11 Protein Identification by Mass Spectrometry Customer Information Package 10 Kinexus Bioinformatics Corporation

11 Please ensure 3 copies of a signed commercial invoice accompany your shipment which specifies your samples are non hazardous and non infectious. Since the samples are not for resale, the value of your shipment should be priced at approximately $1.00 per sample in order to avoid paying additional duties and taxes on entry into Canada. It is highly recommended that customers their courier airway bill number and the date of departure to info@kinexus.ca so we can track your shipment in transit and ensure it arrives in a timely manner. For shipping from outside of North America, we highly recommend send out on a Monday or Tuesday. We will send confirmatory once your shipment arrives at our facility. 8. PRICING INFORMATION Kinexus offers its services at different pricing levels depending on the level of confidentiality required for your samples and whether you are following on from a previous proteomics analysis with our Kinex and/or Kinetworks services. With our full Confidential option, our regular price starts at US $750 per antibody immunoprecipitation and $960 per protein gel slice analyzed by mass spectrometry. At this pricing level, only the species identity needs to be disclosed. With our Non-confidential option, clients can receive a 33% discount on the mass spectrometry analysis price. To receive the 33% discount, Kinexus requires the Client- Supplied Non-Confidential Sample Description Form (NSDF-LY) to be completed in full (Sections A-K) including species, organ, tissue, cell, cell state, fractionation, perturbation, and treatment for each sample being analyzed. Furthermore, if the work is a follow on from previous proteomics studies performed by Kinexus, we offer an additional 20% discount from the Confidential and Non-confidential prices. For volume discounts or quotations for large orders, please contact the Director of Sales & Marketing at KINEXUS (or (Extension 11 or Option 2 on the telephone directory) or sales@kinexus.ca. FOLLOW UP SERVICES AND FORMS 9. FOLLOW UP SERVICES After an immuno-reactive protein with a phospho-site antibody has been identified with our PIMS services, clients may desire to identify the phosphorylation sites and the protein kinases that may target these phosphosites. Kinexus can produce recombinant forms of the immuno-reactive protein or synthesize short peptides that are predicted from the amino acid sequence of the immuno-reactive protein. Using our proprietary Kinase and Phospho-site Predictor algorithms, Kinexus can identify known and putative phospho-sites and the kinases responsible for their phosphorylation. We can test these predictions with our panel of more than 360 human protein kinases. If desired, Kinexus can also help produce specific antibodies against desired phospho-sites. Contact our Customer Services Representative to learn more about our custom proteomics services or visit our website at 10NO11 Protein Identification by Mass Spectrometry Customer Information Package 11 Kinexus Bioinformatics Corporation

12 10. FORMS TO BE COMPLETED All of the forms necessary to use the Protein Identification by Mass Spectrometry (PIMS) services are provided in the Appendices section of this Customer Information Package. Fillable MS-Word versions of some of these forms may be obtained by request by or by phone or directly downloadable from the Kinexus website at Please contact our Technical Service Representatives by at or by phone at Ext. 1 for all enquiries related to technical/research issues, work orders, service fees or request of fillable order forms. All customers are required to complete the following forms for each order placed: A. Kinexus Proteomics Services Agreement - Customers are required to complete and sign our standard Proteomics Services Agreement before their first order can be processed. Unless otherwise specified, this Agreement is valid for all future orders with a standard term of 15 years. B. Service Order Form (PIMS-SOF). The Service Order Form allows us to track all of the various services to be used within an order. C. Service Identification Form (PIMS-SIF). The Service Identification Form permits us to determine which proteomics service screen is to be used for the analysis of two cell/tissue lysate samples together. D. Sample Description Forms Customers should choose one or both of the following forms that is applicable: Non-Confidential Sample Description Form (NSDF-LY); Confidential Sample Description Form (CSDF-LY). The Sample Description Forms (SDF s) allow us to determine the nature of the cell/tissue lysates to be analyzed. One SDF is completed per cell/tissue lysate sample. If clients wish to provide their own antibodies for immunoprecipitation, they should also complete either Non-Confidential Sample Description Forms (NSDF-AB) or Confidential Sample Description Forms (CSDF-AB) as applicable with information of these antibodies. E. Federal Express Airway Bill (if samples are to be delivered by courier). F. Commercial Invoice (required for all customers located outside of Canada). All orders should have as a minimum: 1 SOF, 1 SIF, and 2 SDF forms completed, along with a courier airway bill and commercial invoice. A new Kinexus Service Agreement is not necessary if the client has previously placed an order with Kinexus and submitted a signed Kinexus Service Agreement at that time FOR ALL CUSTOMERS A. Kinexus Service Agreement A Kinexus Service Agreement is required to be signed before the first order 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 us at 10NO11 Protein Identification by Mass Spectrometry Customer Information Package 12 Kinexus Bioinformatics Corporation

13 to request a Microsoft Word version of the document so your requested changes can be made directly into the agreement and ed to us for our final approval. B. Service Order Form (PIMS-SOF) Please ensure: Shipping address and contact name and numbers are specified Billing information is completed as outlined in Section D on the Service Identification Form (PIMS-SIF) 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 Form (PIMS-SIF) For each sample submitted, please ensure the following: In Section A, the customer must assign a unique Client Screen Identification Name to correlate the proteins to be analyzed for each sample submitted In Section B, the type of analysis (Kinex Screen Name) for each sample is specified. For Section C, your sample(s) are identified by completion of Client Supplied Non-Confidential (NSDF-LY) or Confidential (CSDF-LY) Sample Description Forms. Make sure that the Client Screen ID Name in Box A of these forms, matched the Client Screen ID Name in Box A of the PIMS-SIF form In Section D, the level of confidentiality is indicated for correct pricing The form is certified correct and signed and dated D. Sample Description Forms Client-Supplied Non-confidential Sample Description Form for Lysates (NSDF-LY) Client-Supplied Confidential Sample Description Form for Lysates (CSDF-LY) For the cell/tissue lysate samples submitted, please ensure the following: Each sample tube is labeled and properly identified on the form in Section B, including final concentration and volume Your sample is described by completion of Client-Supplied Non-Confidential (NSDF-LY) or Confidential (CSDF-LY) Sample Description Forms by checking the appropriate boxes and entering the appropriate information requested in Sections A-K for Non-confidential samples and Sections A-C for Confidential samples The form is certified correct and signed and dated Note that the information provided on this form will eventually become available to thousands of other scientists in the future with the non-confidentiality pricing. In the spirit of collegiality, please be as accurate as possible in completing the NSDF-LY form in order to not handicap their research efforts should they desire to follow up on your PIMS results. 10NO11 Protein Identification by Mass Spectrometry Customer Information Package 13 Kinexus Bioinformatics Corporation

14 FOR ALL CUSTOMERS SENDING THEIR OWN ANTIBODIES E. Client-Supplied Antibody Description Forms (NSDF-AB and CSDF-AB) For submitted antibodies, please ensure the following: Each antibody sample tube is labeled and properly identified on the form in Section B, including final concentration and volume In Section A, the customer must enter the unique Client Screen Identification Name from Box A of the Service Identification Form (PIMS-SIF) to match the antibody to the lysate to be used for immunoprecipitation with this antibody. The form is certified correct and signed and dated F. Airway bill for Federal Express or any courier that accepts dry ice shipments Complete the airway bill and specify: Priority overnight delivery Bill transportation charges to your institute Place sufficient dry ice to last several days into a Styrofoam shipping container Seal the edges of the Styrofoam container with tape to preserve dry ice longer Dry ice is a hazardous item, so ensure proper labels are attached to the outside of the box; Do not specify Saturday delivery or hold at courier location For Federal Express shipments telephone GO-FEDEX or visit them on-line at or to schedule a pick up or complete your forms For shipments coming from within Canada or the United States, please ship any day from Monday to Wednesday. Do not ship on a Thursday or Friday. For international shipments coming from outside of North America, the best day to ship is on a Monday to ensure arrival in Canada for delivery later the same week It is recommended that customers the date of your shipment and the courier airway bill number with number of samples to Kinexus at info@kinexus.ca to ensure we can track your package should it get held up in Canadian Customs For any customer located outside of Canada, 3 copies of a commercial invoice is required to accompany your shipment (see below) FOR U.S AND INTERNATIONAL CUSTOMER ONLY G. Commercial Invoice (not required by Canadian customers) Please complete the attached commercial invoice with the following information: Date of exportation Shipper/Exporter name, address, phone number Country of export/country of origin 10NO11 Protein Identification by Mass Spectrometry Customer Information Package 14 Kinexus Bioinformatics Corporation

15 Name of courier and the airway bill number Number, type and total weight of package(s) Total declared value of shipment (number of samples x $1.00 per sample) and please specify currency Date, name, signature, and title of authorized person Include three (3) copies of the commercial invoice with the airway bill NOTE: Do not change the value of your shipment to more than $1.00 per sample as this will prompt the custom brokers to charge Kinexus with a duty and GST fee on your package. Since the samples are processed internally and not returned to the customer or resold, there is no real commercial value. The international air waybill is required for all international shipments between Canada and the rest of the world. It is also your customs declaration, which can possibly be used to clear your shipment through customs at the destination. The customs clearance process begins with the description of the air waybill. If the description is too vague or missing, customs authorities may select the shipment for further inspection. All customs paperwork, such as the commercial invoice, must have detailed commodity descriptions. A detailed description on the air waybill and other customs documentation will help speed up the clearance time and reduce your delivery time. In the event that Kinexus must go to a Canada Customs facility to claim the package of samples for client order due to inadequate completion of the commercial invoice, additional charges will apply. 10NO11 Protein Identification by Mass Spectrometry Customer Information Package 15 Kinexus Bioinformatics Corporation

16 SERVICE ORDER FORM PROTEIN IDENTIFICATION BY MASS SPECTROMETRY Subject to terms of the Kinexus Proteomics Services Agreement Form: PIMS-SOF KINEXUS ORDER NUMBER For Kinexus internal use only. CUSTOMER INFORMATION REPEAT CUSTOMER OR NEW CUSTOMER Dr. Mr. Ms. Name of Authorized Representative or Principal Investigator Title/Position Company Name or Institute Department Street Address City State or Province Country Zip or Postal Code Address (Area Code) Telephone Number (Area Code) Facsimile Number Contact Person (if different from Authorized Representative) Address (Area Code) Telephone Number STUDY REPORTS RESULTS SENT BY TO: AUTHORIZED REPRESENTATIVE/INVESTIGATOR AND/OR CONTACT PERSON PRICING INFORMATION Services offered for identification of antibody cross-reactive proteins by immunoprecipitation and mass spectrometry. PRICING - Refer to Section D of the Sample Identification Forms: All prices in U.S. Funds THE PROTEIN IDENTIFICATION IS CARRIED OUT IN TWO STAGES: 1 ST STAGE BY IMMUNOPRECIPITATION OR IMMUNOAFFINITY, 2 ND STAGE BY MASS SPECTROMETRY. Follow on order - One antibody chosen for immunoprecipitation stage of study x US$600 per antibody = $ Follow on order Two or more antibodies chosen for immunoprecipitation stage of study x US$500 per antibody = $ New order - One antibody chosen for immunoprecipitation stage of study x US$750 per antibody = $ New order - Two or more antibodies chosen for immunoprecipitation stage of study x US$625 per antibody = $ Follow on order Non-confidential Number of protein bands for MS identification analysis x US$600 per band = $ Follow on order Confidential Number of protein bands for MS identification analysis x US$800 per band = $ New order Non-confidential Number of protein bands for MS identification analysis x US$720 per band = $ New order - Confidential Number of protein bands for MS identification analysis x US$960 per band = $ Estimated cost of this order (Final price will dependent on number of protein bands actually submitted for mass spectrometry analysis after client approval) Subtotal = $ PROMOTIONAL DISCOUNT IF APPLICABLE (PROVIDE QUOTATION OR REFERENCE NUMBER) - $ Subtotal = $ FOR CANADIAN CUSTOMERS ONLY: Add an additional 12% to the above total for HST (No RT0001): + $ TOTAL COST FOR THIS ORDER TOTAL = $ TOTAL AMOUNT PAYABLE IN U.S FUNDS PAYMENT METHOD PURCHASE ORDER ACCEPTED FROM COMPANIES AND INSTITUTES WITH APPROVED CREDIT. P.O. NUMBER: VISA OR MASTERCARD Print Cardholder Name Visa Number Expires (M/Y) Cardholder Signature BILLING INFORMATION SEND INVOICE TO CUSTOMER AT ABOVE ADDRESS OR SEND INVOICE TO ACCOUNTS PAYABLE CONTACT: Dr Mr Ms Accounts Payable Contact Name Company Name or Institute Street Address City State or Province Country Zip or Postal Code (Area Code) Telephone Number AUTHORIZATION CUSTOMER HAS READ THE KINEXUS PROTEOMICS SERVICES AGREEMENT AND AGREES TO BE BOUND BY THE TERMS AND CONDITIONS: Print Name of Authorized Representative or Principal Investigator Authorized Signature Date (Y/M/D) How did you originally hear about the PIMS services? Direct Mail Web Site Advertisement Referral Conference or Trade Show Other

17 PROTEIN IDENTIFICATION BY MASS SPECTROMETRY SERVICE IDENTIFICATION FORM Subject to terms of the Kinexus Service Agreement Form: PIMS-SIF KINEXUS ORDER NUMBER NAME: (Authorized Representative or Principal Investigator) COMPANY/INSTITUTE: PROTEIN IDENTIFICATION BY MASS SPECTROMETRY SERVICE REQUESTED: (WITH CLIENT LYSATES) Clients have the option of choosing antibodies provided by Kinexus or their own antibodies, or any combination thereof. Please refer to visit the Kinexus website at to access the complete list of current antibodies available from Kinexus. For each selected antibody, provide the appropriate ID codes from the Kinexus Antibody List as well as the names of the protein targets (and phospho-sites sites if applicable). Clients may also provide their own antibodies for this service if they fully disclose on accompanying NSDF-AB forms the nature of the probing antibodies (including immunogen sequence, the animal species in which the antibody was produced as well as manufacturer s name and catalogue number if it is commercially sourced). SERVICE REQUESTED: PROTEIN IDENTIFICATION BY MS 1st stage - Immunoprecipitation 2nd stage - Mass spectrometry analysis 4 to 10 mg protein in a cell or tissue lysate is normally required for each immunoprecipitation KINEXUS ID NUMBER (Bar Code Identification Number) For Kinexus Internal Use Only. A. CLIENT SCREEN ID NAME: Customer ID: Provide ID name of your choice for your reference and for use in Box A of Client-Supplied Sample Description (NSDF-LY, CSDF-LY) and Client- Supplied Antibody Description (NSDF-AB, CSDF-AB) forms. B. ANTIBODY SELECTION: For Kinexus supplied antibodies, record the appropriate ID codes from the Kinexus Antibody List downloadable from To reduce the risk of error, please also provide the abbreviated name of the protein target of the antibody and, if applicable, the desired phosphorylation site. If you are using this service to follow up on previous Kinex or Kinetworks data, please use the ID Code provided with the results of your previous order or contact our Technical Services Representatives. If clients wish to include antibodies of their own, please provide the client name for each antibody that has been entered into Box B from the completed and attached Client-Supplied Antibody Description (NSDF-AB, CSDF-AB) forms. Ab 1. Kinexus ID Code: Protein name: Phospho-site: Client supplied. Client name for antibody Ab 2. Kinexus ID Code: Protein name: Phospho-site: Client supplied. Client name for antibody Ab 3. Kinexus ID Code: Protein name: Phospho-site: Client supplied. Client name for antibody Ab 4. Kinexus ID Code: Protein name: Phospho-site: Client supplied. Client name for antibody Ab 5. Kinexus ID Code: Protein name: Phospho-site: Client supplied. Client name for antibody Ab 6. Kinexus ID Code: Protein name: Phospho-site: Client supplied. Client name for antibody Ab 7. Kinexus ID Code: Protein name: Phospho-site: Client supplied. Client name for antibody Ab 8. Kinexus ID Code: Protein name: Phospho-site: Client supplied. Client name for antibody C. SAMPLE IDENTIFICATION: For a client supplied sample, please complete a Client- Supplied Non-confidential Sample Description Form (NSDF- LY) or a Client-Supplied Confidential Sample Description Form (CSDF-LY). If this cell/tissue lysate sample has been analyzed before on a Kinex KAM antibody microarray, please also provide the previous Kinexus Order Number in order to qualify for a 20% discount off of our regular pricing. It is important if you are using this service to follow up on previous Kinex or Kinetworks data, that you use the ID Codes in Section B that were provided with the results of your previous order or you should contact our Technical Services Representatives. Kinexus Order Number Previous Kinex Analysis: Previous Kinetworks Analysis: D. PRICING: Immunoprecipitation (IP) Stage: Follow on from previous Kinex or Kinetworks order* Single antibody US$600 each Two or more antibody IP US$500 each New order Single antibody US$750 each Two or more antibody IP US$625 each Mass Spectrometry Analysis Stage: Follow on from previous Kinex or Kinetworks order* US$600 each US$800 each New order US$720 each US$960 each * Box C must be completed with the Kinexus Order Number of the previous Kinex KAM antibody microarray analysis or Kinetworks multi-immunoblot analysis. Use this pricing information for completion and submission of Service Order Form: PIMS-SOF. Name of person completing this form Signature Date (Y/M/D)

18 FOR LYSATES CLIENT SUPPLIED NON-CONFIDENTIAL SAMPLE DESCRIPTION FORM Subject to terms of the Kinexus Service Agreement Form: NSDF-LY KINEXUS ORDER NUMBER NAME: (Authorized Representative or Principal Investigator) COMPANY/INSTITUTE: Non-Confidential Service Requested and Lysate Sample Details: Please refer to the Customer Information Package for the particular Kinexus proteomics service that you are requesting for details on how to prepare and ship your lysates to Kinexus for testing. Clients are required to complete all Sections A-K to qualify for the Non-Confidential pricing level for the Kinexus Proteomics Services if they provide their own lysates for analysis. If sample details are to remain Confidential, please complete instead the Client-Supplied Confidential Sample Description Form (CSDF-LY) in Sections A-C. If you need further assistance, please contact a technical service representative by calling toll free in North America KINEXUS ( ) or by at info@kinexus.ca. Please check the appropriate tick boxes. A. CLIENT SCREEN ID NAME + KINEXUS SERVICES NAME: CLIENT ID: KINEXUS PROTEOMICS SERVICES NAME: Use the Client ID Name that you entered in Box B on the Service Identification Form (SIF). The Kinexus Proteomics Services abbreviated name should be used from the SIF. C. SPECIES: Human (Homo sapiens) Sex: Male Female M/F pooled Unknown Rat (Rattus norvegicus) # Animals: Age: Weight: Mouse (Mus musculus) Other Provide scientific & common name: E. TISSUES: A. Organ source of tissue: B. Tissue name: C. Disease condition if appropriate: B. SAMPLE IDENTIFICATION: Client Name for Sample: Control: Yes No Concentration (mg/ml): KINEXUS ID NUMBER (Bar Code Identification Number) D. SAMPLE SOURCE: Volume (µl): (FOR INTERNAL USE ONLY) Tissues: Yes No If yes, proceed to Section E Cells: Yes No If yes, proceed to Section F F. CELLS: Is your sample a primary culture? Yes No Is your sample an established cell line? Yes No A. Name of cell line: B. Organ source of cells: C. Tissue or cell type: D. Disease condition if appropriate: G. CELL STATE: N/A Subconfluent Quiescent Confluent Scenescent Proliferating Apoptosing Differentiated Stressed H. FRACTIONATION: Detergent-solubilized total lysate Cytosolic (Soluble) Particulate (Detergent-solubilized) IP - If yes, indicate antibody or ligand used: Other purification: I. PERTURBATION: Normal untreated If yes, proceed to Section K Normal treated If yes, proceed to Section J Diseased untreated If yes, proceed to Section K Diseased treated If yes, proceed to Section J J. TREATMENTS: Please indicated if you used combined [CMB] or sequential [SEQ] treatments and provide details on your treatment: 1. Name of compound/stimuli: Concentration: Time: CMB SEQ 2. Name of compound/stimuli: Concentration: Time: CMB SEQ 3. Name of compound/stimuli: Concentration: Time: CMB SEQ Details of treatment: K. ADDITIONAL SAMPLE INFORMATION: Please include any additional information that differentiates your samples: Transgenic: Yes No Knockout: Yes No Wildtype: Yes No Transfected/Over-expressed: Yes No Mutant: Yes No If you answered yes to any of the above, please specify details including if there was any deprivation (such as serum/growth factor/drug/site of mutation) prior to treatment: I hereby certify that all the sample information provided in this order is correct and accurate to the best of my knowledge. To qualify for the non-confidential pricing level, I agree that all Sections A-K must be completed in full otherwise the confidential pricing level will be applied. I further acknowledge that I may be contacted by a Kinexus representative for additional information if any section is unclear. Name of person completing this form Signature Date (y/m/d)

19 FOR LYSATES CLIENT SUPPLIED CONFIDENTIAL SAMPLE DESCRIPTION FORM Subject to terms of the Kinexus Service Agreement Form: CSDF-LY KINEXUS ORDER NUMBER NAME: (Authorized Representative or Principal Investigator) COMPANY/INSTITUTE: Confidential Service Requested and Lysate Sample Details: Please refer to the Customer Information Package for the particular Kinexus proteomics service that you are requesting for details on how to prepare and ship your lysates to Kinexus for testing. Clients are required to complete Sections A-C for the Confidential pricing level for Kinexus Proteomics Services if they provide their own lysates for analysis. Note that a Confidential analysis is performed at a higher pricing level than a Non-Confidential analysis. Clients should instead complete all of Sections A-C on the Client-Supplied Non-Confidential Sample Description Form (NSDF-LY) to qualify for the non-confidential pricing. To obtain further assistance, please contact a technical service representative by calling toll free in North America KINEXUS ( ) or by at info@kinexus.ca. Please check the appropriate tick boxes. A. CLIENT SCREEN ID NAME + KINEXUS SERVICES NAME: CLIENT ID: KINEXUS PROTEOMICS SERVICES NAME: Use the Client ID Name that you entered in Box B on the Service Identification Form (SIF). The Kinexus Proteomics Services abbreviated name should be used from the SIF. B. SAMPLE IDENTIFICATION: Client Name for Sample: Control: Yes No Concentration (mg/ml): Volume (µl): C. SPECIES: Human (Homo sapiens) Sex: Male Female M/F pooled Unknown Rat (Rattus norvegicus) # Animals: Age: Weight: Mouse (Mus musculus) Other Provide scientific & common name: KINEXUS ID NUMBER (Bar Code Identification Number) (FOR INTERNAL USE ONLY) A. CLIENT SCREEN ID NAME + KINEXUS SERVICES NAME: B. SAMPLE IDENTIFICATION: CLIENT ID: KINEXUS PROTEOMICS SERVICES NAME: Use the Client ID Name that you entered in Box B on the Service Identification Form (SIF). The Kinexus Proteomics Services abbreviated name should be used from the SIF. Client Name for Sample: Control: Yes No Concentration (mg/ml): Volume (µl): C. SPECIES: Human (Homo sapiens) Sex: Male Female M/F pooled Unknown Rat (Rattus norvegicus) # Animals: Age: Weight: Mouse (Mus musculus) Other Provide scientific & common name: KINEXUS ID NUMBER (Bar Code Identification Number) (FOR INTERNAL USE ONLY) A. CLIENT SCREEN ID NAME + KINEXUS SERVICES NAME: B. SAMPLE IDENTIFICATION: CLIENT ID: KINEXUS PROTEOMICS SERVICES NAME: Use the Client ID Name that you entered in Box B on the Service Identification Form (SIF). The Kinexus Proteomics Services abbreviated name should be used from the SIF. Client Name for Sample: Control: Yes No Concentration (mg/ml): Volume (µl): C. SPECIES: Human (Homo sapiens) Sex: Male Female M/F pooled Unknown Rat (Rattus norvegicus) # Animals: Age: Weight: Mouse (Mus musculus) Other Provide scientific & common name: KINEXUS ID NUMBER (Bar Code Identification Number) (FOR INTERNAL USE ONLY) I hereby certify that all the sample information provided in this order is correct and accurate to the best of my knowledge. I further acknowledge that I may be contacted by a Kinexus representative for additional information if any section is unclear. Name of person completing this form Signature Date (y/m/d)

20 FOR ANTIBODIES CLIENT-SUPPLIED NON-CONFIDENTIAL SAMPLE DESCRIPTION FORM Subject to terms of the Kinexus Proteomics Services Agreement Form: NSDF-AB KINEXUS ORDER NUMBER NAME: (Authorized Representative or Principal Investigator) COMPANY/INSTITUTE: Non-Confidential Service Requested and Antibody Sample Details: Please refer to the Customer Information Package for the particular Kinexus proteomics service that you are requesting for details on how to prepare and ship your antibodies to Kinexus for testing. For Non-Confidential pricing you must fully describe the nature of the probing antibodies (including immunogen sequence, the animal species in which the antibody was produced as well as manufacturer s name and catalogue number if it is commercially sourced). Please note that in the event that clients do not wish to disclose the source or nature of the antibodies that they are providing, then Confidential Pricing must apply. Clients must still complete Sections A to C for Confidential analyses. Please check the appropriate tick boxes. If you need assistance completing this form, contact a technical service representative by calling toll free in North America KINEXUS ( ) or by at info@kinexus.ca. A. CLIENT SCREEN ID NAME + KINEXUS SERVICES NAME: CLIENT ID: KINEXUS PROTEOMICS SERVICES NAME: Use the Client ID Name that you entered in Box B on the Service Identification Form (SIF). The Kinexus Proteomics Services abbreviated name should be used from the SIF. C. SPECIES OF ANTIBODY ORIGIN AND TYPE: Rabbit Monoclonal Mouse Goat Human Other Provide common name: E. IMMUNOGEN INFORMATION: Polyclonal Species of origin of protein or peptide sequence: B. ANTIBODY IDENTIFICATION: Client Name for Antibody: Concentration (mg/ml): Volume (µl): Recommended dilution for immunoblotting: Clients should provide at least enough antibody for making 3 ml of antibody solution at the desired titre per microarray. We recommend 30 µl of 1 mg/ml antibody. KINEXUS ID NUMBER (Bar Code Identification Number) (FOR INTERNAL USE ONLY) D. COMMERCIAL SOURCE OF ANTIBODY (if applicable) Supplier Name: Supplier Catalog Number: Supplier Lot Number: F. AMINO ACID SEQUENCE OF IMMUNOGEN Protein: Yes Protein Name: Peptide: Yes If yes, please go to Box F and provide the amino acid sequence of the immunizing peptide if it is known A. CLIENT SCREEN ID NAME: CLIENT ID: Use the Client ID Name that you entered in Box B on the Service Identification Form (SIF). The Kinexus Proteomics Services abbreviated name should be used from the SIF. C. SPECIES OF ANTIBODY ORIGIN AND TYPE: Rabbit Monoclonal Mouse Goat Human Other Provide common name: E. IMMUNOGEN INFORMATION: Polyclonal Species of origin of protein or peptide sequence: B. ANTIBODY IDENTIFICATION: Client Name for Antibody: Concentration (mg/ml): Volume (µl): Recommended dilution for immunoblotting: Clients should provide at least enough antibody for making 3 ml of antibody solution at the desired titre per microarray. We recommend 30 µl of 1 mg/ml solution of antibody. KINEXUS ID NUMBER (Bar Code Identification Number) (FOR INTERNAL USE ONLY) D. COMMERCIAL SOURCE OF ANTIBODY (if applicable) Supplier Name: Supplier Catalog Number: Supplier Lot Number: F. AMINO ACID SEQUENCE OF IMMUNOGEN Protein: Yes Protein Name: Peptide: Yes If yes, please go to Box F and provide the amino acid sequence of the immunizing peptide if it is known I hereby certify that all of the information about antibodies that I provided in this order is correct and accurate to the best of my knowledge. Name of person completing this form Signature Date (y/m/d)