BTI Study

Posted on January 11 2010 by

BTI began genetic research of the various Brain Types in the mid-1990’s. Neurotransmitter DNA analysis was done at a prominent American university. Though neurotransmitter evaluation is still ongoing, BTI has also ventured into other genetic fields, searching for additional clues. One such area is Proteomics, where our ultimate goal is to identify the various inborn Brain Types strictly by urine analysis.

Due to a few people who do not want us to succeed, we’ve kept much of our research confidential. We will unveil it at the appropriate time. In the meantime, however, BTI will provide some basic examples of our research. With this in mind, we offer a portion of a Brain Type and Proteomic analysis.

OPTIMIZATION PROTOCOL

Study Title: Proteomic Patterns and Brain types
Goal: To identify proteomic patterns that relate to Brain Types using Ciphergen SELDI-TOF.

I. Introduction

The Brain Type Institute has identified different kinds of brain types which have been used to classify behaviors (cognitive, motor, and visual) associated with success in various sports and skill positions within sports.

The creation of an accurate cellular protein profile from an individual’s serum can yield important identification information that can be used as fingerprint patterns to distinguish different brain typings. This may contribute in skill and behavior selection amongst chosen candidates.

SELDI technology enables selective protein retention on protein chip array surfaces by means of distinct chromatographic chemistries. Once a peak of interest has been detected, further techniques can be applied to enrich or purify the peak through a combination of column and on-chip purification strategies. Patterns generated by protein chip reader mass spectrometric means are analyzed by a data-mining tool which will lead to important identification information.

II. Objective and Plan

Brain typing may be reflected by differences in serum proteomic patterns. To test this hypothesis, (proprietary) Research Lab will use bioinformatics tools to analyze serum proteomic mass spectra from subjects with known brain types to identify patterns that can be used to accurately classify specimens by brain type.

III. Serum Samples

1. Serum has been derived from 6 individuals who have signed an IRB consent form allowing the use of their specimens for assay development. The serum was collected under an IRB-approved protocol (“p”01) that allows for use of remnant, de-identified or anonymized human subject samples for in vitro analysis under the guidelines defining Exemption from Human Subject Research as defined by the Office of Human Research Protection.

2. Test System

2.1. Ciphergen SELDI_TOF Instrument

2.2. Strong Anion Exchange Chip (SAX2) and Weak Cation Exchange Chip (WCX2), these chips are used for test optimization

3. Procedures

3.1.1. Individual Serum stored in freezer is thawed to room temperature

3.1.2. Serum protein is measured by coomassie dye binding assay to obtain 1ug of protein concentration. (Bradford Method PRO:QUNT).

3.1.3. Each serum sample is diluted with binding buffer1:20 fold specific for observation of protein peaks on a SAX2 and WCX2 chip.

3.1.4. Each sample was washed with the same binding buffer. The washes varied with the surface chemistry of the chip.

3.1.5. Large number of differentially displayed proteins for each serum sample is obtained.

4. Serum preparation Procedure

4.1.1. Load 250 ul of binding buffer to each spot on the chip from A to E. Incubate on high frequency shaker at room temperature for 5 minutes. Do not allow buffer to air-dry.

4.1.2. Prepare the serum by adding 10 ul of each serum sample into 250 ul of fresh SAX2 and WCX2 binding buffer.

4.1.3. Load the entire 250 ul of each diluted serum sample including the control

4.1.4. 0044, 0045, 0048, 0049, 0052 and 0057 into spot A to E on both chips.

4.1.5. Place the chip in a bioprocessor. Attach the top securely.

4.1.6. Mix at 250 rpm on a shaker for two hours.

4.1.7. Remove samples from SAX2 and WCX2 chips.

4.1.8. Wash the chips.

4.1.9. Wash each spot with 250ul of binding buffer five times, followed by washing twice with 250ul water.

4.1.10. Wipe dry around spots, Add 0.5 ul of sinapinic acid EAM, to each spot, and air-dry.

4.1.11. Analyze chip using the protein chip system.

4.1.12. The manufacturer’s instructions will be followed for Instrument Operation.

4.1.13. Specimens will be analyzed using the SELDI TOF chip reader.

5. Analysis

Analysis result of SELDI-TOF for serum samples 0044,0045,0048,0049,0052 and 0057

Method is applied for profiling a population of protein in a given serum sample according to the size and net electrical charge of the individual proteins. The chart below is the readout of spectrum of peaks. The position of an individual protein in the spectrum corresponds to its “time of flight”, because of small proteins fly faster and the large proteins fly more slowly. In SELDI the protein is pre selected allowing selected protein to bind to the treated surface of a metal bar, which is coated with a specific chemical that binds a subset of proteins with in the serum sample. SAX2 chip utilized in this experiment is chosen for proteins with low pI’s. WCX2 chip is chosen for proteins with high pI’s both used for biomarker discovery coated with strong anion and weak cationic exchange to analyze molecules that have negative and positive charges on the surface. Active spots for SAX2 contain cationic, quaternary ammonium groups that interact with the negative charges on the surface of target proteins, e.g. aspartic acid or glutamic acid. Active spots for WCX2 contain weak anionic, carboxylate groups that interact with the positive charges on the surface of target proteins, e.g. lysine, arginine or histidine.

Proteomic Pattern Result

The discriminating pattern formed by a small key subset of proteins or peptides buried among the entire repertoire of thousands of proteins represent in the sample spectrum can distinguish between serum of this selected group of BT patients.

The comparison of the six spectrums shown in fig 3 and 4 shows no discriminatory patterns defined by the peaks between these six selected group of BT patients on SAX2 and WCX2 protein chips.

Elevated levels of certain protein is observed in 0044,0045 and 0048 patients with WCX2 chip.

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