INTEGRATED FLUORESCENCE-LABELING AND POLYACRYLAMIDE GEL ELECTROPHORESIS FOR ANALYSIS OF PROTEIN ISOFORMS Akwasi Apori and Amy E. Herr University of California, Berkeley, USA

ABSTRACT We present a new native protein analysis format that automates and integrates all assay steps required for fluorescence-based isoform analysis. Our assay combines salient features of on-chip PAGE (polyacrylamide gel electrophoresis) with well- controlled protein labeling within an on-chip ‘nanoreactor’ defined by a polyacrylamide size-exclusion membrane. The method uses efficient, addressable electrophoretic transport to seamlessly integrate: native protein labeling, background signal reduction, protein enrichment, and native PAGE protein analysis. Results demonstrate native PAGE separations of protein isoforms in less than 2 minutes after protein enrichment and labeling at the membrane.

KEYWORDS: electrophoresis, isoform, fluorescence labeling, polyacrylamide

BACKGROUND While integration of multiple functions is a hallmark of microfluidic tools, efforts to fully incorporate sample preparation would benefit from a surge in technology development. Specifically, innovation is needed for tools that allow dynamically- adjustable protein enrichment, fractionation of complex samples based on physicochemical properties, and efficient mixing of reagents with enriched sample fractions. As part of the needed on-chip preparatory tool repertoire, protein labeling with fluorescent dye is essential to realize the “sample-to-answer” paradigm of many lab-on-a-chip systems. While we demonstrate an approach that promises to provide many of these needed features, we focus on characterization of on-chip protein labeling and subsequent native PAGE analysis.

A handful of notable efforts in on-chip labeling have been reported [1,2]; nevertheless, simplified on-chip labeling techniques promise to expand the protein analysis application space. Here we detail development of technology that maximizes protein labeling, while minimizing background signal from unconjugated dye molecules. Previous efforts by our group and colleagues have utilized size- exclusion membranes fabricated using in situ photopolymerization for enrichment and mixing of a priori labeled proteins and immunoreagents [3,4]. Advancing on this prior work, we introduce a streamlined technique that utilizes the small pore-size polyacrylamide membranes to yield an on-line ‘nanoreactor’ defined by the volume of fluid in front of the membrane. The size-exclusion properties of the membrane allow elimination of background fluorescence from small dye molecules and provide a confined geometry for well-controlled protein-dye interactions. Subsequent electrophoretic protein analysis occurs rapidly in a contiguous polyacrylamide gel. To demonstrate this as-of-yet unreported work, we utilize the technique for analysis

Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences October 12 - 16, 2008, San Diego, California, USA

978-0-9798064-1-4/µTAS2008/$20©2008CBMS 15 of protein isoforms – a first step towards studying important disease-relevant co- and post-translational protein modifications (i.e., glycosylation).

We utilize small pore-size polyacrylamide gel membranes (15-22%T, 3.6-6%C) to: 1.) enrich protein samples at the membrane interface using purely electrophoretic flow, 2.) co-localize non-covalent dye with the enriched protein sample in an aqueous environment, and 3.) subsequently elute and analyze labeled proteins using on-chip polyacrylamide gel electrophoresis (PAGE; 6%T, 3.6%C). Figure 1 provides a schematic overview of the technique. Key features of the assay design include: i) filter-based reduction of background fluorescence arising from unconjugated dye molecules, ii) dynamic protein enrichment, iii) well-controlled and adjustable protein-dye ratios (using timed electrophoretic loading of each species), and iv) use of a confined geometry at the membrane interface to actively co-locate protein and dye during labeling incubation.

Figure 1. Size exclusion membrane allows on-chip protein labeling & subsequent native PAGE analysis. (left) Image of 100 um size-exclusion membrane. (right figures) An electric current is used to drive proteins from the sample well to enrich at the membrane. Non-fluorescing dye is driven to label proteins at membrane and, upon interaction, the protein-fluorophore complex fluoresces. Native PAGE separation commences.

RESULTS We utilize a dye (Quant-iT, Invitrogen) that fluoresces after interacting with protein (fluorogenic). Figure 2 shows non-fluorescent dye loading against and interacting with protein enriched at the membrane. Our initial experiments suggest that a minimum level of detectable fluorescence can be identified after 50 seconds of labeling the enriched proteins at the membrane (experiment not shown).

Figure 2. Image sequence shows on-chip labeling of unlabeled native proteins . (Initial concentration: 126 μM BSA and 7 μM Urease, high concentrations used for imaging purposes).

Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences October 12 - 16, 2008, San Diego, California, USA 16 Figure 3 shows results for near-baseline native PAGE analysis of a protein sample. After on-chip labeling, native PAGE isoform separations were complete in < 2 minutes generating distinguishable fluorescence peaks. We are currently optimizing the native PAGE conditions to analyze disease-relevant isoforms in complex fluids, as a “walk away” platform for high-throughput targeted proteomics.

Figure 3. Proteins are separated post on-chip labeling. 1) unbound dye (Quant-It) 2) BSA (66kDa) and 3) mass isoforms of Urease (272 kDa, 545 kDa)

SUMMARY & FUTURE WORK In this work, we present a facile method for protein analysis that automates and integrates native protein labeling, background signal reduction, protein enrichment, reagent mixing, and native PAGE protein analysis. On-going work centers on optimizing the labeling conditions for both native PAGE and protein sizing assays.

ACKNOWLEDGEMENTS The authors would like to acknowledge the University of California, Berkeley (AEH ) and the National Defense Science and Engineering Graduate Fellowship (AA) for financial support.

REFERENCES [1] L.J. Jin, B. C. Giordano, J.P. Landers, Anal. Chem. 73(20): 4994-4999 (2001). [2] L. Bousse, S. Mouradian, A. Minalla, H. Yee, K. Williams, R. Dubrow, Anal. Chem. 73(6): 1207-1212 (2001). [3] A.E. Herr, A.V. Hatch, D.J. Throckmorton, H.M. Trans, J.S. Brennan, W.V. Giannobile, and A.K. Singh, PNAS, 104, 5268-5273 (2007). [4] A.V. Hatch, A.E. Herr, D.J. Throckmorton, J.S. Brennan, and A.K. Singh, Anal. Chem. 78(14): 4976-4984 (2006).

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