On-Column Sample Enrichment for Capillary Electrophoresis Sheathless Electrospray Ionization Mass Spectrometry: Evaluation for Peptide Analysis and Protein Identification George M. Janini,* ,² Ming Zhou, ² Li-Rong Yu, ² Josip Blonder, ² Michelle Gignac, ‡ Thomas P. Conrads, ² Haleem J. Issaq, ² and Timothy D. Veenstra ² Laboratory of Proteomics and Analytical Technologies and Image Analysis Laboratory, SAIC-Frederick, Inc., National Cancer Institute at Frederick, P.O. Box B, Frederick, Maryland 21702 Although several designs have been advanced for coupling sample enrichment devices to a sheathless electrospray ionization-mass spectrometry (MS) interface on a capillary electrophoresis (CE) column, most of these approaches suffer from difficulties in fabrication, and the CE separa- tion efficiency is degraded as a result of the presence of coupling sleeves. We have developed a design that offers significant improvements in terms of ease of fabrication, durability, and maintenance of the integrity of the CE- separated analyte zones. Capillaries with different inside and outside diameters were evaluated to optimize the performance of the CE-MS system, resulting in a mass limit of detection of 500 amol for tandem MS analysis of a standard peptide using a 20-μm-i.d. capillary. The improved design incorporates an efficient method to preconcentrate a sample directly within the CE capillary followed by its electrophoretic separation and detection using a true zero dead-volume sheathless CE-MS inter- face. Testing of this novel CE-MS system showed its ability to characterize proteomic samples such as protein digests, in-gel-digested proteins, and hydrophobic pep- tides as well as to quantitate ICAT-labeled peptides. Capillary electrophoresis (CE) is well known for its ability to provide highly selective separations. Its direct coupling with mass spectrometry (MS) has shown great potential in proteomic analysis as demonstrated by many laboratories 1-22 and detailed in several reviews. 23-26 CE-MS has many advantages compared to the more widely used technique of liquid chromatography (LC) -MS, including separation efficiency, speed, and simplicity. Since the CE capillary is constructed using open tubular fused silica, contamination due to carry-over effects from separation to separation is minimized. The time between consecutive CE experiments is much shorter than LC-based separations with gradient elution, since the CE capillary column does not require extensive reequilibration between analyses. On the negative side, however, CE has generally lagged behind LC in terms of ruggedness, sample capacity, and detection sensitivity. While attomole mass limit of detection (MLOD) have been reported in several CE-MS applications, 1,2,4,8,16,20,21 the concentra- tion limit of detection (CLOD) using conventional CE-MS capillaries is in the micromolar range compared to the nanomolar range for LC-MS. This disparity is mainly a result of the large sample volume capacity of a capillary LC column (i.e., microliters) compared to a CE capillary (i.e., nanoliters). This loading capacity limitation hampers the utility of CE-MS for the analysis of typical biological samples, where proteins and peptides are often present at submicromolar concentrations. In addition, most commercial CE-MS systems use coaxial sheath flow that lowers the overall * To whom correspondence should be addressed. Phone: 301-846-7189. Fax: 301-846-6037. E-mail: Janini@ mail.ncifcrf.gov. † Laboratory of Proteomics and Analytical Technologies. ‡ Image Analysis Laboratory. (1) Janini, G. M.; Conrads, T. P.; Wilkens, K. L.; Issaq, H. J.; Veenstra, T. D. Anal. 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Chem. 2003, 75, 5984-5993 5984 Analytical Chemistry, Vol. 75, No. 21, November 1, 2003 10.1021/ac0301548 CCC: $25.00 © 2003 American Chemical Society Published on Web 09/19/2003