Microdeposition Device Interfacing Capillary Electrochromatography and Microcolumn Liquid Chromatography with Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Tony J. Tegeler, Yehia Mechref, Kirk Boraas, James P. Reilly, and Milos V. Novotny* Department of Chemistry, Indiana University, Bloomington, Indiana 47405 A sample deposition device has been constructed and optimized for interfacing CEC and capillary LC columns to MALDI mass spectrometry. For CEC analysis, the device is composed of an inlet buffer reservoir and an outlet buffer reservoir connected to a matrix reservoir through a connection sleeve. The matrix reservoir is connected to a deposition capillary via another connection sleeve. CEC eluent is transported to the matrix reservoir via a capillary that is connected to the deposition capillary by the connection sleeve inside the matrix reservoir. This connection sleeve also acts as a mixing chamber, allowing the CEC eluent to be mixed with matrix prior to deposi- tion. Complex glycan mixtures can be separated by CEC using hydrophilic-phase monolithic columns, with capil- lary eluent being deposited on a standard MALDI plate along with a suitable matrix solution. Thousands of discrete, highly homogeneous dots can be generated for a subsequent mass spectrometric analysis. With minor modifications, this device is also applicable to capillary LC of peptides using gradient elution. In this configura- tion, the outlet of the LC column is connected to a deposition capillary inside a matrix reservoir through a connection sleeve that allows mixing of the LC effluent with an appropriate matrix. The device has been evaluated with the tryptic digests of proteins. The current and future investigations of biological processes are likely to demand increased sophistication of both separation methodologies and mass spectrometry (MS). Proteome complex- ity is particularly evident in mammalian systems where posttrans- lational modifications of proteins abound, creating numerous protein forms that may originate distantly from a single gene. Protein glycosylation is a significant posttranslational modification of highly organized and sophisticated eukaryotic systems. In contrast with simple protein modifications, the structural analysis of glycoproteins and their detection in complex biological mixtures may demand the best proteomic 1-5 and glycomic methodologies. 6-8 Whether the separation of proteins from complex mixtures is accomplished through a time-honored approach of two-dimen- sional (2-D) gel electrophoresis or one of the recently investigated 2-D solution alternatives (for a review, see ref 9), the separated fractions are typically digested to peptides prior to their MS investigations. For a preselected pool of glycoproteins, a more labor-intensive approach is involved, which includes protease digestion into mixtures of peptides and glycopeptides and a release of complex glycans from the glycopeptides that all must be separated and structurally identified. Both electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) are widely used in conjunction with MS for analyzing both peptide mixtures and oligosaccharide pools. A miniaturized ESI source has a distinct advantage of procedural simplicity in capillary LC-based combinations with MS. However, this is less true with capillary electromigration tech- niques in which the separation systems must be electrically insulated. 10-13 With all ESI-based investigations, the aliquot is entirely consumed in one LC/MS analysis. Consequently, MS/ MS data can be lost during a rapid on-the-fly selection of precursor ions or due to detector saturation. Depending on the nature and complexity of investigated proteins, this may decrease the number of identified components and their sequence coverage. At present, capillary LC/MS using nanospray ESI is widespread in proteomic laboratories, while capillary electrochromatography (CEC) using a similar type of ESI interface has recently been introduced into the investigation of complex glycan samples. 14-16 * Corresponding author. E-mail: novotny@indiana.edu. Phone: (812) 855- 4532. Fax: (812) 855-8300. (1) Saraf, A.; Yates, J. R., III. Protein Arrays, Biochips, Proteomics 2003, 233- 253. (2) Giometti, C. S. Adv. Protein Chem. 2003, 65, 353-369. (3) Issaq, H. J. Adv. Protein Chem. 2003, 65, 249-269. (4) Naylor, S.; Kumar, R. Adv. Protein Chem. 2003, 65, 217-248. (5) Smith, R. D.; Anderson, G. A.; Lipton, M. S.; Masselon, C.; Pasa-Tolic, L.; Udseth, H.; Belov, M.; Shen, Y.; Veenstra, T. D. Adv. Protein Chem. 2003, 65, 85-131. (6) Reinhold: V. N.; Reinhold: B. B.; Costello, C. E. Anal. 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Chem. 2004, 76, 6698-6706 6698 Analytical Chemistry, Vol. 76, No. 22, November 15, 2004 10.1021/ac049341b CCC: $27.50 © 2004 American Chemical Society Published on Web 10/16/2004