We demonstrate a strategy of maximizing the performance of reversed-phase (RP) liquid chromatography (LC) tandem mass spectrometry (MS/MS) for efficient shotgun proteome analysis by optimizing the sample loading to the instrument in an off-line two-dimensional (2D) LC tandem MS platform. To determine the quantity of peptides present in a proteome digest or fractionated peptides from strong-cation exchange (SCX) separation, an automated system based on RPLC with a rapid step solvent gradient for peptide elution and ultraviolet (UV) detection was developed. This system also allowed the purification of the peptides by removing salts and other impurities present in a sample. It was found that controlling the amount of peptides injected into a RPLC MS/MS system was critical to achieve the maximum efficiency in peptide and protein identification. With the use of off-line 2D-LC-MS/MS, peptide fractions from the first dimension of separation were desalted and quantified, followed by injecting the optimal amount of the sample into RPLC-MS/MS for peptide sequencing. The application of this strategy was demonstrated in the proteome profiling of breast cancer MCF-7 cells. From the analysis of 28 SCX fractions with each injecting 1 microg of sample into a 75 mum x 100 mm C18 column interfaced to a quadrupole/time-of-flight mass spectrometer, a total of 2362 unique proteins or protein groups were identified with a false positive peptide identification rate of 0.19%, as determined by target-decoy proteome sequence searches. Replicate 2 h runs of individual fractions with the exclusion of precursor ions of peptides already identified in the first runs resulted in the identification of an additional 549 unique proteins or protein groups with a false positive identification rate of 0.60%. This example illustrated that off-line 2D-LC-MS/MS, with maximal sample injection to the RPLC-MS, is an effective method for shotgun proteome analysis. Finally, the advantages and limitations of this method, compared to other methods, are discussed.

Monolithic silica capillary columns were prepared by a sol-gel process in fused-silica capillaries with an inner diameter of 50 microm and were modified by coating of cellulose tris(3,5-dimethylphenylcarbamate). Influences of the factors in the modification process on enantiomer separations were investigated. The prepared columns were used to perform enantiomer separations by CEC. Fifteen and two pairs of enantiomers were separated under aqueous and nonaqueous mobile phases, respectively, and most of them were baseline-separated with very high column efficiencies. The Van Deemter curve was found flat under high linear velocity of the mobile phase, which indicated favorable kinetic properties of the prepared columns. Baseline separation of a pair of enantiomers was achieved in 90 s with high-column efficiency by short-end separation under high voltage.

A capillary electrochromatography (CEC) monolithic column with zwitterionic stationary phases was prepared by in situ polymerization of butyl methacrylate, ethylene dimethacrylate, methacrylic acid, and 2-(dimethyl amino) ethyl methacrylate in the presence of porogens. The stationary phases have zwitterionic functional groups, that is, both tertiary amine and acrylic acid groups, so the ionization of those groups on the zwitterionic stationary phase was affected by the pH values of the mobile phase, and further affects the strength and direction of the electroosmotic flow (EOF). Separations of alkylbenzenes and polycylic aromatic hydrocarbons based on the hydrophobic mechanism were obtained. Separation of various types of polar compounds, including phenols, anilines, and peptides, on the prepared column were performed under CEC mode with anodic and cathodic EOF, and different separation selectivities of those polar analytes were observed on the monolithic capillary column by using mobile phases with different pH values.Ionizable groups on the surface of the stationary phase are necessary to generate substantial electoosmotic flow (EOF) for capillary electrochromatography (CEC). 1 Strictly speaking, most stationary phases used in CEC are ion-exchangers. Silica-based packing materials, which have been most widely used in CEC and can be regarded as weak cation exchangers, generate cathodic EOF due to the ionization of the residual silanol groups on the surface of packings. Recently, strong ion exchangers and so-called mixed-mode packing materials consisting of ionic groups such as sulfonic acids or quaternary amines and hydrocarbon chains have attracted much attention in CEC, because these packings ensure stable EOF over an extended pH range. 2 The direction of the EOF depends on the charges on the surface of the stationary phases, so stationary phases with positively charged functional groups, such as amino groups or ammonium groups, generate an EOF from cathode to anode, whereas stationary phases carrying negatively charged groups, such as sulfonic acid and carboxylic acid, generate cathodic EOF. To date, the vast majority of reports on CEC concern EOF with one direction, that is, cathodic or anodic EOF, for a CEC column; however, zwitterionic stationary phases, which can make it possible to generate EOF with different directions for one column, are seldom investigated in CEC. In fact, there are many reports concerning chromatographic separation with zwitterionically modified materials, although it is still one of the new liquid chromatography separation modes studied in recent years. [3][4][5][6] Hu et al. 7 established the zwitterionic functionality via dynamically adsorbing the ODS (octadecyl silica) column with a sulfobetaine-type zwitterionic surfactant by the interaction between the C 18 groups on the surface and the hydrophobic tail of the surfactant. To overcome the drawback of inferior stability due to loss of functional moieties from the dynamically attached layer on the surface of stationary phases, ...