Low-Attomole Electrospray Ionization MS and MS/MS Analysis of Protein Tryptic Digests Using 20-μm-i.d. Polystyrene -Divinylbenzene Monolithic Capillary Columns Alexander R. Ivanov, Li Zang, and Barry L. Karger* Barnett Institute and Department of Chemistry, Northeastern University, Boston, Massachusetts 02115 This work explores the use of 20-μm-i.d. polymeric polystyrene-divinylbenzene monolithic nanocapillary col- umns for the LC-ESI-MS analysis of tryptic digest peptide mixtures. In contrast to the packing of microparticles, capillary columns were prepared, without the need of high pressure, in fused-silica capillaries, by thermally induced in situ copolymerization of styrene and divinylbenzene. The polymerization conditions and mobile-phase compo- sition were optimized for chromatographic performance leading to efficiencies over 1 0 0 0 0 0 plates/ m for peptide separations. High mass sensitivity (∼1 0 amol of peptides) in the MS and MS/ MS modes using an ion trap MS was found, a factor of up to 2 0 -fold improvement over 7 5 - μm- i.d. nanocolumns. A wide linear dynamic range (∼4 orders of magnitude) was achieved, and good run-to-run and column-to-column reproducibility of isocratic and gradient elution separations were found. As samples, both model proteins and tissue extracts were employed. Gradi- ent nano-LC-MS analysis of a proteolytic digest of a tissue extract, equivalent to a sample size of ∼1000 cells injected, is presented. The high mass sensitivity identification and quantitation of large numbers of peptides from protein digests is a major goal in proteomics. Nanoflow liquid chromatography, using commercially available 75- and 100- µm-i.d. reversed-phase columns, offers the advantages of high resolution, high mass sensitivity, and low sample and mobile-phase consumption. However, analysis of a limited amount of sample (e.g., laser capture microdissected cells, immunoprecipitated proteins, 2-D gel spots, etc.) can still be challenging with the above columns. For a fixed limited amount of sample injected, columns with smaller inner diameter can decrease chromatographic band dilution 1-3 and thus increase the signal for concentration-sensitive ESI-MS. 4 However, narrow-bore columns (particularly less than 50- µm i.d.) are difficult to pack with conventional microparticles because of the very high pressure required to overcome the low column permeability. 4-6 Monolithic nanocapillary columns of 20- µm i.d. or less can be a good alternative to microparticle-packed columns because of their relative ease of manufacture, without the need of high pressure, and their high-performance characteristics. 7-13 Relative to packed columns, porous monolithic nanocapillary columns also result in potentially less clogging of the ESI tip and do not require frits. Several approaches for synthesis of polymeric monoliths in capillaries of 100-300- µm i.d. have been published in which the reaction mixture is placed in the capillary, followed by UV or thermally induced in situ polymerization. 7-9,11-14 High efficiencies of the narrow-bore capillary columns are found, due to decreased flow dispersion and a homogeneous packing bed structure. The bulk liquid flow in ultranarrow-i.d. capillary columns is reduced by 1 order of magnitude (15-50 nL/ min), relative to 75-100- µm- i.d. columns, which results in analytes being dissolved in much lower eluent volume with lower amount of ionic and neutral species of the mobile phase in the chromatographic band. 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