(34b) On-Chip Protein Extraction and Albumin Exclusion From Plasma and Serum Using Isotachophoresis

harp counter migrating
bands. (Bottomng by ion by using 

ON-CHIP
PROTEIN EXTRACTION AND ALBUMIN EXCLUSION FROM PLASMA AND SERUM USING
ISOTACHOPHORESIS

Yatian Qu, ¹ Lewis A.
Marshall, ² Juan G. Santiago¹

¹
Department of Mechanical Engineering, Stanford University

² Department
of Chemical Engineering, Stanford University

Stanford, CA 94305, USA

Human
blood plasma and serum contain thousands of proteins, which makes them ideal matrices
for diagnostics and the discovery of disease-related biomarkers.
ADDIN EN.CITE
<EndNote><Cite><Author>Colantonio</Author><Year>2005</Year><RecNum>4</RecNum><DisplayText><style Article">17</ref-type><contributors><authors><author>Colantonio,
D. A.</author><author>Dunkinson,
C.</author><author>Bovenkamp, D. E.</author><author>Van
Eyk, J.
E.</author></authors></contributors><auth-address>Department
of Physiology, Queen&apos;s University, Kingston, Ontario, Canada.</auth-address><titles><title>Effective
removal of albumin from
serum</title><secondary-title>Proteomics</secondary-title><alt-title>Proteomics</alt-title></titles><periodical><full-title>Proteomics</full-title><abbr-1>Proteomics</abbr-1></periodical><alt-periodical><full-title>Proteomics</full-title><abbr-1>Proteomics</abbr-1></alt-periodical><pages>3831-5</pages><volume>5</volume><number>15</number><keywords><keyword>Albumins/*isolation
&amp; purification</keyword><keyword>Ammonium Sulfate</keyword><keyword>Biological
Markers/blood</keyword><keyword>Blood Protein
Electrophoresis/*methods</keyword><keyword>Chemical
Fractionation</keyword><keyword>Chemical
Precipitation</keyword><keyword>Chromatography,
Affinity</keyword><keyword>Electrophoresis, Gel, Two-Dimensional</keyword><keyword>Electrophoresis,
Polyacrylamide
Gel</keyword><keyword>Female</keyword><keyword>Humans</keyword><keyword>Male</keyword><keyword>Serum/*chemistry</keyword><keyword>Triazines</keyword></keywords><dates><year>2005</year><pub-dates><date>Oct</date></pub-dates></dates><isbn>1615-9853
(Print)&#xD;1615-9853
(Linking)</isbn><accession-num>16145708</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/pubmed/16145708</url></related-urls></urls><electronic-resource-num>10.1002/pmic.200401235</electronic-resource-num></record></Cite></EndNote>¹ However,
the concentrations of these proteins vary over 10 orders of magnitude. ADDIN EN.CITE  ADDIN
EN.CITE.DATA 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This large dynamic
range hinders the detection of the low-abundance proteins that are more likely
to be potential biomarkers.
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Albumin,
the most abundant protein in plasma, constitutes 50-90% of the total protein mass
but carries little analytical information. ADDIN EN.CITE
<EndNote><Cite><Author>Chen</Author><Year>2005</Year><RecNum>2</RecNum><DisplayText><style Y.</author><author>Lin, S. Y.</author><author>Yeh,
Y. Y.</author><author>Hsiao, H. H.</author><author>Wu,
C. Y.</author><author>Chen, S. T.</author><author>Wang,
A.
H.</author></authors></contributors><auth-address>Core
Facilities for Proteomic Research, Taipei,
Taiwan.</auth-address><titles><title>A modified protein
precipitation procedure for efficient removal of albumin from
serum</title><secondary-title>Electrophoresis</secondary-title><alt-title>Electrophoresis</alt-title></titles><periodical><full-title>Electrophoresis</full-title><abbr-1>Electrophoresis</abbr-1></periodical><alt-periodical><full-title>Electrophoresis</full-title><abbr-1>Electrophoresis</abbr-1></alt-periodical><pages>2117-27</pages><volume>26</volume><number>11</number><keywords><keyword>Acetone</keyword><keyword>Animals</keyword><keyword>Blood
Protein Electrophoresis/methods</keyword><keyword>Blood
Proteins/*isolation &amp; purification</keyword><keyword>Chemical
Fractionation</keyword><keyword>Chemical
Precipitation</keyword><keyword>Electrophoresis, Polyacrylamide
Gel</keyword><keyword>Humans</keyword><keyword>Methods</keyword><keyword>Mice</keyword><keyword>Proteomics/*methods</keyword><keyword>Serum
Albumin/*isolation &amp;
purification</keyword><keyword>Trichloroacetic
Acid</keyword></keywords><dates><year>2005</year><pub-dates><date>Jun</date></pub-dates></dates><isbn>0173-0835
(Print)&#xD;0173-0835 (Linking)</isbn><accession-num>15880626</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/pubmed/15880626</url></related-urls></urls><electronic-resource-num>10.1002/elps.200410381</electronic-resource-num></record></Cite></EndNote>⁴ Therefore,
extracting low abundance proteins from plasma or serum while depleting albumin
is an essential sample preparation procedure in proteomics. Here, we report on the development of a novel
on-chip system and protocol for protein extraction and exclusion of albumin from
human plasma or serum samples. This method can
effectively and quickly purify proteins and deplete albumin with little
manual labor, and it can also be automated and integrated with
microfluidics-based proteomic assays.

We perform this protein extraction and albumin exclusion
technique by using isotachophoresis (ITP). ITP achieves selective, rapid pre-concentration and
separation of molecules based on their electrophoretic mobility. ADDIN
EN.CITE <EndNote><Cite><Author>Persat</Author><Year>2009</Year><RecNum>5</RecNum><DisplayText><style Nucleic Acids from Whole Blood Using Isotachophoresis</title><secondary-title>Analytical
Chemistry</secondary-title></titles><periodical><full-title>Analytical
Chemistry</full-title></periodical><pages>9507-9511</pages><volume>81</volume><number>22</number><dates><year>2009</year></dates><urls></urls></record></Cite></EndNote>⁵ Analytes are focused
between relatively high mobility leading electrolyte (LE) ions and low mobility
trailing electrolyte (TE) ions.  We take advantage of the relatively low isoelectric point (pI)
of human serum albumin (pI of 4.7-4.8),
which is lower than the pI of the majority of plasma
or serum proteins. We operate ITP at a pH such that the proteins with pI higher than albumin's will be
positively charged, allowing them to be focused in cationic ITP.  Albumin, along with proteins with pI lower than albumin's, remains unfocused in channel.

We
have designed a large-volume microfluidic chip as shown in Figure 1 to
demonstrate this technique. This chip is fabricated from polystyrene and
includes two branch channels connecting to an air outlet, forming a T-junction
in the middle. The channels are 1mm wide and 150 µm deep, with a volume of 14
µL for each branch. To perform extraction experiments, we load plasma or serum
sample to one branch and LE to the other branch by applying vacuum through air
outlet. The LE branch has an elution reservoir, where the proteins
can be collected off the chip. The elution reservoir is also connected to a
buffering reservoir, which can provide additional buffering capacity.

We have successfully extracted proteins and eliminated albumin from 1 µL plasma and serum samples by using
this technique. We monitored
extracted protein zone by imaging spiked yellow fluorescent proteins (YFP). We
verified albumin exclusion by imaging spiked bovine serum albumin (BSA) labeled
with fluorescent dye, Alexa Fluor 647. We demonstrated
that non-selective extraction and selective extraction of proteins could be
achieved by using different buffer systems, as shown in Figure2.  To analyze extracted proteins, we
performed sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie Blue staining (Figure 3).
We compared the extracted proteins to unprocessed plasma or serum proteins. We
recovered at least 10 protein bands from a range of molecular weight. As we
expected, albumin is barely visible from the selective extracted samples. Protein band absorbance analysis on SDS-PAGE gel indicated ~90% albumin depletion efficiency.

Figure 1. Microfluidics chip
design for large-volume protein extraction and albumin exclusion
from human plasma or serum. Each reservoir holds approximately 30
µL liquid.
Each branch channel has a volume of 14 µL.

Figure 2. Comparison of non-selective and selective
ITP protein extraction chemistries: (Top) Beta-alanine was used as TE. Both YFP and albumin
focused into the ITP zone. (Bottom) 6-aminocaproic acid was used as TE, and a band of YFP focused
under ITP while albumin was excluded.

Figure 3. Off-chip SDS-PAGE
separation images of proteins extracted with ITP and from control serum samples
where proteins were not extracted with ITP. Albumin is the most abundant
protein band present in the raw serum sample (middle). However, the proteins
extracted from serum using ITP (right) contain no albumin band, while
maintaining the other protein bands from the original serum.

Figure 4. Protein absorbance
analysis on SDS-PAGE gel.  Shown are repetitions of albumin amount
relative to transferrin amounts. We used the transferrin band as a reference to
normalize the absorbance of albumin band for both extracted proteins samples
via ITP and unprocessed serum samples. The absorbance ratio indicates ~90%
albumin depletion efficiency.

REFERENCES

 ADDIN EN.REFLIST 1.  Colantonio,
D. A.; Dunkinson, C.; Bovenkamp, D. E.; Van Eyk, J. E., Effective removal of
albumin from serum. Proteomics 2005, 5 (15), 3831-5.

2.  Granger, J.; Siddiqui, J.; Copeland, S.;
Remick, D., Albumin depletion of human plasma also removes low abundance
proteins including the cytokines. Proteomics
2005, 5 (18), 4713-8.

3.  Echan, L. A.; Tang, H. Y.; Ali-Khan, N.;
Lee, K.; Speicher, D. W., Depletion of multiple high-abundance proteins
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4.  Chen, Y. Y.; Lin, S. Y.; Yeh, Y. Y.;
Hsiao, H. H.; Wu, C. Y.; Chen, S. T.; Wang, A. H., A modified protein precipitation
procedure for efficient removal of albumin from serum. Electrophoresis 2005, 26 (11), 2117-27.

5.  Persat, A.; Marshall, L. A.; Santiago,
J. G., PuriÞcation of Nucleic Acids from Whole Blood Using Isotachophoresis. Analytical Chemistry 2009, 81 (22), 9507-9511.