Present plasma biomarker discovery increasingly demands high-throughput plasma sample processing. To discover clinically-relevant biomarkers, throughput has to increase to a level of 100 sample processing per day. One of the most important rate-limiting steps in sample processing is depletion of highly-abundant proteins (HAPs). Seppro®Tip platform allows simultaneous processing of 12 samples at a time in a completely automated fashion. Up to 100 plasma or serum samples can be processed daily using the system.

Product Features
High throughput. Up to 100 samples/day.
Complete automation. No hands-on manipulations.
Cost-effective. Microtips are regenerated automatically and
can be re-used for 30 runs.
Buffers come in disposable cartridges, which are loaded
for each run.
Highly effective: >96% removal of high-abundant proteins
from human serum/plasma samples.

Specifications
15 µl of plasma sample/run
12 samples per run processing
65 minutes per cycle
Each tip can be used 30 times
Each tip is automatically regenerated for next cycle

Types of Media
IgY12 (removal of top 12 HAPs from human and primate plasma/serum
samples)
IgY-C7 (removal of top 7 HAPs from cattle plasma/serum samples)
IgY-R7 (removal of top 7 HAPs from rat plasma/serum samples)
IgY-M7 (removal of top 7 HAPs from mouse plasma/serum samples)
IgY-D10 (removal of top 10 HAPs from canine plasma/serum samples)
IgY-Rubisco (removal of Rubisco protein from plant protein mixtures)

The plasma samples were subjected to IgY12 HAP removal using consecutive Seppro®Tip runs. Processed samples were subjected to 2D-fluorescent gel analysis
(Fig. 3 and 4) and mass spectometry analysis (Fig. 5). A total of 50 runs were conducted. Figure 3 shows tip-to-tip variability, while Figure 4 shows run-to-run variability on 2D-gels. During the first 30 runs tips were removing top 12 HAPs in a highly-consistent and completely reproducible fashion; the final 20 runs showed some variation in run-to-run HAP removal efficiency.

Figure 3. HAP Depletion Efficiency –Tip-to-Tip Variability
50 µg of protein eluted in the wash fraction following elution of depleted protein was
labeled with 400 pmol of Cy2, Cy3, Cy5 dye (Seppro®Tip 3.4.5, respectively). IPG pH 4-7, 24 cm strips were used in the first dimension 8-16% SDS-PAGE was used for the second dimension. Shown here are two-channel comparisons. Areas appearing in either red or green are differentially abundant between the two shown runs; areas in yellow are of similar abundance in both runs. Aliquots of the same serum control
sample HPM0351B1 were used.

Figure 4. HAP Depletion Efficiency –Run-to-Run Variability (Run 35)
Run 35. 50 µg of protein eluted in the wash fraction following elution of depleted protein was labeled with 400 pmol of Cy2, Cy3, Cy5 dye (Seppro®Tip 3, 4, 5,respectively). IPG pH 4- 7, 24 cm strips were used in the first dimension 8-16% SDS-PAGE was used for the second dimension. Shown here are two-channel comparisons. Areas appearing in either red or green are differentially abundant between the two shown runs; areas in yellow are of similar abundance in both runs.
Aliquots of the same serum control sample HPM0351B1 were used.

Figure 5 shows mass spectrometry data on run-to-run variability of sample processing. It is clear that run-to-run variability is negligible.

Conclusions
Studies show that the Seppro®Tip immunoaffinity fractionation
system reproducibly removes the top 12 abundant proteins in an
automated and high-throughput fashion. It removes over 90% of
the total plasma protein mass from a sample.

The reproducibility of Seppro®Tip is consistent, with low variability
between tips in a single run and between runs.

Seppro®Tip can be recycled for at least 30 uses.

This system brings biomarker discovery to a new level. It eliminates
the bottleneck of plasma sample preparation, permits highthroughput
processing of thousands of samples, thus accelerating
biomarker discovery and validation.

Seppro®Tip is the only available system for high-throughput serum
or plasma sample processing for biomarker discovery that can be
used in conjunction with mass spectrometry and 2D-gel analysis
methods.

12 µg of IgY
Seppro®Tip depleted protein was digested
with trypsin and
profiled using
MALDI-TOF-MS.

Seppro®Tip3,
Run 2

Samples were spotted on MALDI target in quadruplicate to illustrate the
reproducibility of both
pattern and intensity.

Seppro®Tip3, Run 3
Figure 5. MALDI-TOF
data on Run-to-Run
variability.
Demonstrates
reproducibility between
Run #2 and Run #3