Shen Hu 1 Jiang Jiang 2 Lillian M. Cook 2 Dawn P. Richards 2 Laura Horlick 2 Brandon Wong 2 Norman J. Dovichi 1 1 Department of Chemistry, University of Washington, Seattle, WA, USA 2 Department of Chemistry, University of Alberta, Edmonton, AB, Canada Capillary sodium dodecyl sulfate-DALT electrophoresis with laser-induced fluorescence detection for size-based analysis of proteins in human colon cancer cells Capillary sodium dodecyl sulfate (SDS)-DALTelectrophoresis (SDS-DALT-CE) refers to CE separation of proteins based on their size; DALT is the abbreviation for Dalton, the unit used to describe molecular weight. In this work, seven proteins from 18 to 116 kDa were denatured by SDS, labeled by 3-(2-furoyl) quinoline-2-carboxaldehyde, sepa- rated by SDS-DALT-CE in polyethylene oxide sieving matrix, and detected by laser- induced fluorescence (LIF) in a sheath flow cuvette. This method was combined with detergent differential fractionation, which is a protein fractionation method using a se- ries of detergent-containing buffers to sequentially extract protein fractions from cells, to analyze the proteins in HT29 human colon adenocarcinoma cells. In addition, on- column labeling was demonstrated for protein analysis by SDS-DALT-CE with LIF, and applied to analysis of proteins in a single HT29 cancer cell. Most proteins had molecu- lar masses from 10 to 120 kDa. Similar protein profiles were obtained for single cells and protein extract of a large cell population. Keywords: Capillary sodium dodecyl sulfate-DALTelectrophoresis / Detergent differential fractio- nation / Human cancer cells / Laser-induced fluorescence / Single cell protein analysis EL 5096 1 Introduction Capillary SDS-DALT electrophoresis (SDS-DALT-CE), a size-based CE separation method for proteins [1], has several advantages over classical SDS – polyacrylamide gel electrophoresis (SDS-PAGE). These advantages include ease of automation, high analysis speed, small sample volume, as well as accurate quantitation of pro- teins and determination of their molecular weights. Cur- rently, linear or slightly branched polymers, such as linear polyacrylamide, polyethylene oxide (PEO), polyethylene glycol, dextran, and pullulan, are often used as the sieving matrix for SDS-DALT-CE analysis ofproteins [2–8]. Com- paring with cross-linked polyacrylamide gel matrix [9, 10], these polymers add great flexibility to SDS-DALT-CE method because they are water-soluble and replaceable between CE experiments. Although UV absorbance is a common detector in SDS- DALT-CE, it produces low sensitivity for proteins due to the short optical pathlength across the capillary. However, many proteins, especially some regulatory proteins, are expressed at very low level in cells [11, 12]. Highly sensi- tive detection technologies such as laser-induced fluores- cence (LIF) are needed to characterize such low-abun- dance proteins. In particular, we are interested in develop- ing a technology to allow the study of protein expression in single somatic cells. Currently, LIF is the most common detector for CE analysis of proteins in single cells [13–20]. Detergent differential fractionation (DDF) is a method used for sequential extraction of proteins from cells or tis- sues [21–23]. By using a series of detergent-containing buffers including digitonin/EDTA, Triton X-100/EDTA, Tween 40/deoxycholate (DOC), and cytoskeletal solubili- zation buffer, DDF reproducibly yields four distinct frac- tions, which contain cytosolic proteins and soluble cyto- skeletal elements, membrane and organelle proteins, nuclear membrane and soluble nuclear proteins, and detergent-resistant cytoskeletal filaments with nuclear matrix proteins [23]. DDF is simple, reproducible, and ultracentrifuge-independent. It does not damage the structure or function of the subcellular compartments and can enrich low-abundance species, and the tech- nique has many applications [21, 24]. In this work, a size-based separation method by SDS- DALT-CE with LIF is described for analysis of proteins. This method is combined with DDF to characterize the pro- teins expressed in HT29 human colon cancer cells. On- column labeling is also demonstrated for protein analysis by SDS-DALT-CE with LIF. Finally, this method is applied to analysis of proteins in a single HT29 cancer cell. Correspondence: Professor Norman J. Dovichi, Department of Chemistry, University of Washington, Seattle, WA 98195-1700, USA E-mail: dovichi@chem.washington.edu Fax: 1206-685-8665 Abbreviations: DDF , detergent differential fractionation; DOC, deoxycholate; FQ, 3-(2-furoyl)quinoline-2-carboxalde- hyde; PGO, polyethylene oxide 3136 Electrophoresis 2002, 23, 3136–3142  2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0173-0835/02/1809–3136 $17.501.50/0