Protein Lysate Microarrays

Clay ScottRyan McConnellShannon Neeley

Biological and Technical Background

Motivation

DNA/RNA microarrays are used to determine gene expression

Proteomic profiling can help yield more direct answers to biological questions

Those molecules that can answer our questions are proteins, not mRNA

– Biological effector molecules– Diagnostic markers– Pharmaceutical targets

History of Proteomic Profiling

2D-PAGE (two-dimensional polyacrylamide gel electrophoresis)– Introduced in 1975– Semiquantitation of most abundant 1000 spots– Problems with identifying spots with a particular protein

Microarray formats for proteomic profiling– More recent development (papers published in 2001)– Robotic spotting of antibodies that capture the protein

molecule to be assessed

Reverse-phase protein lysate microarrays

Opposite configuration from previous microarray

Samples assessed are robotically spotted An antibody is used to measure amount of a

particular protein present in the sample Limitation: Measure one protein per slide Advantage: All samples analyzed side-by-side

in a single array – Can compare protein levels across samples rather

than samples across protein type

Protein lysate preparation

Lysis: The dissolution or destruction of cells by the action of a specific lysin that disrupts the cell membrane

Lysate: The cellular debris and fluid produced by lysis

Lysis Method Description ApparatusWaring BlenderPolytronDounce HomogenizerPotter-Elvehjem HomogenizerFrench Press

Sonication SonicatorHigh frequency sound waves shear cells

Freeze/ThawFreezer or dry ice/ethanol

Repeated cycles of freezing and thawing disrupt cells through ice crystal formation

Manual grinding Mortar and pestleGrinding plant tissue, frozen in liquid nitrogen

Table 1. Techniques used for the physical disruption of cells.

MechanicalRotating blades grind and disperse cells and tissues

Liquid Homogenization

Cell or tissue suspensions are sheared by forcing them through a narrow space

Protein Lysate Array Design

Each microarray is a glass slide containing particular protein samples from different patients

18 spots for every sample– 3 replicates– 6 dilution levels (with

dilution factor of 2)

Western Blotting—used to screen specificity of antibodies

Choose Antibody that will bind to protein

Detection of protein on microarray

The slide is exposed to the antibody Antibody binds to the protein, depending on

how much protein is present Microarray is scanned to form an image with

darker spots reflecting higher levels of protein Use two antibody detection system

Statistical Applications

From Image to Number

Software draws a circle around each spot

Darker spots have greater quantities of protein

sVOL reflects the total amount of protein

sVOL = (πr2)[(average intensity inside circle)-(average background intensity)]

Two-fold serial dilutions

The Data Set 80 x 6 x 3 matrix of sVOL values for the caspase protein 80 patients on one microarray chip 6 two-fold serial dilutions for each patient 3 replicates for each patient Control serum data set for calibration and error-assessment purposes

DilutionsP

A

T

I

E

N

T

S

1

80

6

Potential Tasks to Undertake

1. From the 18 sVOL values for each sample, extract a single robust number that is representative of that sample

° for example, the mean is not robust

2. Provide an error estimate of that number

Saturation DetectionDevise procedure for extracting linear portion of log(sVOL)

For highly concentrated samples, circles drawn by computer may be too small (sVOL smaller than expected)

For large dilutions, sVOL is dominated by background noise (sVOL tails off)

Fine Tuning the Segmentation Program

Outlying sVOL values may occur even when saturation is not a problem

Detecting outliers may help improve the segmentation software