1 2 RPPA applications present two common primary needs for users to - PDF document

1

2

RPPA applications present two common primary needs for users to obtain robust data….. The first need is the required SENSITIVITY to detect low abundance proteins from LOW EXPRESSION LEVEL SAMPLES or from LABILE SAMPLES OF VERY SMALL MASS…. This has driven many users to adopt SIGNAL AMPLIFICATION methods to achieve detection of the pathways they seek to observe, with either Colorimetric or Fluorescent enhanced sensitivity techniques. The SECOND user need for ROBUST RPPA data is DATA ACCURACY provided by CONFIDENT NORMALIZATION METHODS and BROAD DYNAMIC RANGE. As we’ll see later, this ACCURACY is optimally provided by INTRA-SPOT MULTICOLOR multiplexed methods and this CANNOT BE PROVIDED by these current techniques using amplification. The ArrayCAM system presented here achieves BOTH the SENSITIVITY to detect low abundance protein and provides ROBUST multi-color intra-spot data normalization. 3

The Grace Bio-Labs ArrayCAM Proteomic Profiling System is a suite of these complimentary components SPECIFICALLY DESIGNED and DEVELOPED to function TOGETHER providing optimal assay performance 4

Obtaining sensitive measurements of low abundance proteins begins with immobilizing as much protein on the array substrate as possible and Porous nitrocellulose provides the highest protein binding capacity among microarray surfaces. PNC non-covalently binds protein, retaining TERTIARY and QUATERNARY structures preserving BINDING SITES and ANTIGENICITY. This quantifies to 500X GREATER BINDING CAPACITY yielding HIGHEST S/N translating into 50X LOWER LOD’s. 5

Porous Nitrocellulose is optimal when used in combination with Fluorescent detection modalities due to emission and excitation light RESONANT SCATTERING AMPLIFICATION propagated by the virtue of optimized pore size and structure harmonized to wavelengths. This provides a 150 fold signal enhancement when compared to identical quantities of bound probe to other surfaces. 6

When employing fluorescent detection the inherent native autofluorescence of PNC must be also considered. At visible light emission wavelengths considerable auto-fluorescence is observed, however migrating detection wavelengths to the near IR range mitigates the presence of autofluorescence. In fact, moving from emission wavelengths of 635 nm to 800 nm, we observe a 4X greater S/N. 7

To achieve optimal fluorescence in the near IR range with PNC, we employ a Quantum Nanoparticle for assay detection. The quantum nanoparticle (QNP) is a PROTEIN SIZED FLUORESCENT INORGANIC CRYSTAL which is excited at a single wavelength while emitting very NARROW EMISSION bands, facilitating robust multi-color multiplexing. 40X Brighter than other organic fluorophores, these molecules are also PHOTO-STABLE, and won’t degrade or quench under REPEATED EXCITATION or due to other normal environmental conditions providing an ARCHIVABLE RECORD of assay data. 8

The high binding capacity of PNC requires extremely effective blocking and requires specifically developed reagent sets for optimal results As seen here, use of optimized blocking and assay reagents provides improved S/N and sensitivity. Super G blocks very strongly and can over-block for some applications. QBlock is a specially formulated non-protein blocker optimized for fluorescent applications. 9

RELIABLE IMAGING is a critical facet to confident RPPA results and is one of the most EXPENSIVE COMPONENTS of RPPA assay systems. Combining the unique SINGLE EXCITATION wavelength properties of QNC we have developed a LOW COST MULTIWAVELENGTH ARRAY IMAGING INSTRUMENT which provides complete slide images in under 1 minute. ArrayCAM contains four emission filters, and is typically equipped with 3 Near IR and one Colorimetric channel. It is COMPACT and is offered in a BENCH-TOP or miniaturized version appendable to robotic high throughput liquid handling systems. 10

To demonstrate the ArrayCAM imaging capabilities, we benchmarked against a GenePix confocal laser scanner. When observing slide dilution series imaged on both instruments, the ArrayCAM provides identical signal-background intensities to that of the more expensive and complex GenePix confocal laser scanner. 11

Finally, the ArrayCAM employs an extremely rapid easy to use automated software package to expedite spot intensity measurements, eliminating manual alignment processes of typical software. Here is a quick video to demonstrate the speed of the automated array image analysis, First you SELECT a TEMPLATE based on the commonly used GAL file. Next, select the features you wish MEASURED and REPORTED as well as the background and intensity analysis methods. OPEN the IMAGE Then click to START the ANALYSIS Here the software speeds through 640 spots in less than 1 minute, measuring, size, location, backgrounds and intensities Then derived template can be confirmed, and adjusted if needed Then the data is ready to review. 12

The accuracy of the ArrayCAM automated spot finding software is compared here with that of automated spot finding of the GenePix software. Both automated spot finding systems are benchmarked against spot intensities obtained by manual spot location and analysis on the same slide. As seen HERE, Automated ArrayCAM software exhibited nearly perfect correlation to the manual benchmark method where the comparable Automated GenePix method, HERE deviated greatly from the benchmark manual method. The ArrayCAM results requires no additional manual template adjustments greatly reducing image analysis time to obtain concordance with the correct spot intensities. 13

This system as a whole enables us to reliably multiplex multiple detection colors within an array without any cross-talk or signal bleed over. In this example, we’ve probed cell lysates with three different species antibodies and then simultaneously detected with three anti-species detection antibodies at wavelength channels, 800, 655 and 585 nm. Observing the control spots in the center of the arrays, we can see that there is essentially no cross reactivity or bleed over occurring between these three detection channels. 14

Application of intra-spot multiplexing provides increased assay throughput, and a very robust and reliable method of data normalization for assay standardization and increased accuracy. In this example of throughput using arrays containing Raf 1 kinase inhibitor time course treated C32 cell lysates we tracked on the same slide total and phospho proteins normalized with GAPDH. (Call out blue ERK and green S6, showing total protein and pERK stable, where pS6 responding strongly over the time course.) 15

Employing one of three channels for normalization with GAPDH provides a method for adjusting gross assay signal levels to compensate for variations in total deposited protein sample during arraying, cell or tissue health or basic assay variability. Measuring pERK inter-spot cell lysate signal consistencies across these slides we compared different methods of normalization. Four methods included signal normalization using measurements of either total protein or GAPDH from a separate slide, un-normalized data and lastly intra-spot multi-color multi-plexed normalization with GAPDH. Intra-spot normalization provided the best data consistency, reducing %CV 10 – 25% over non-normalized data and 5-15% over other external slide normalization methods. This is due to normalization measurements performed on the exact same spot as the pERK as opposed to another separate slide. 16

Fluorescent multi-color multi-plexing is not a new technique and LI-COR offers organic Near IR dyes for this application. Using the same Raf 1 kinase inhibitor time course treated C32 cell lysates arrays we compared our QNC with ArrayCAM method to the optimized LI-COR method imaged with a two-laser confocal scanner. The results showed the QNC and low-cost ArrayCAM provided equivalent results to the methods using the far more expensive and complex two-laser near IR scanner. In many cases, as seen here, we observed superior sensitivity with the QNC and ArrayCAM, yielding much greater resolution of these changes in protein expressions over these time- course studies. 17

Yesterday we heard Ginny Espinosa present findings using two color QNC and ArrayCAM to measure intra-spot GAPDH normalized breast cancer pathway markers benchmarked against amplified colorimetric detection systems using a slide auto-stainer. We were interested to observe the absolute sensitivities between the QNC and colorimetric methods so we compared LOD of GAPDH on a titration series of NIH 3T3 and JURKAT cell lysates between three experimental methods. QNC ArrayCAM method performed both on the autostainer at GMU and on the benchtop at Grace Bio-Labs and the amplified colorimetric system at GMU. We found very similar LOD’s demonstrating QNC provides equivalent or better sensitivity without the need for expensive and time consuming amplification methods. 18

We also observed sensitivity with low affinity primary antibody tests with QNC where a 1:20 dilution was required to achieve equivalent or better LOD than the colorimetric results on the auto-stainer. We felt this was a large amount of primary antibody and observing experiments using manual bench-top methods and including agitation, we could observe equivalent performance to the colorimetric and QNC 1:20 auto-stainer methods using a much lower 1:200 primary antibody dilution. 19