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. 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This large dynamic range hinders the detection of the low-abundance proteins that are more likely to be potential biomarkers. ADDIN EN.CITE  ADDIN EN.CITE.DATA 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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

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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 improves protein profiling capacities of human serum and plasma. Proteomics 2005, 5 (13), 3292-303.

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.