Two-dimensional liquid chromatography is often used to reduce the proteomic sample complexity prior to tandem mass spectrometry analysis. The 2D-LC performance depends on the peak capacity in both chromatographic dimensions, and separation orthogonality. The peak capacity and selectivity of many LC modes for peptides is not well known, and mathematical characterization for orthogonality is underdeveloped. Consequently, it is difficult to estimate the performance of 2D-LC for peptide separation. The goal of this paper was to investigate a selectivity of common LC modes and to identify the 2D-LC systems with a useful orthogonality. A geometric approach for orthogonality description was developed and applied for estimation of a practical peak 2D-LC capacity. Selected LC modes including various RP, SCX, SEC, and HILIC were combined in 2D-LC setups. SCX-RP, HILIC-RP, and RP-RP 2D systems were found to provide suitable orthogonality. The RP-RP system (employing significantly different pH in both RP separation dimensions) had the highest practical peak capacity of 2D-LC systems investigated.

Two-dimensional high performance liquid chromatography is a useful tool for proteome analysis, providing a greater peak capacity than single-dimensional LC. The most popular 2D-HPLC approach used today for proteomic research combines strong cation exchange and reversed-phase HPLC. We have evaluated an alternative mode for 2D-HPLC of peptides, employing reversed-phase columns in both separation dimensions. The orthogonality of 2D separation was investigated for selected types of RP stationary phases, ion-pairing agents and mobile phase pH. The pH appears to have the most significant impact on the RP-LC separation selectivity; the greatest orthogonality was achieved for the system with C18 columns using pH 10 in the first and pH 2.6 in the second LC dimension. Separation was performed in off-line mode with partial fraction evaporation. The achievable peak capacity in RP-RP-HPLC and overall performance compares favorably to SCX-RP-HPLC and holds promise for proteomic analysis.

This paper presents an improved analytical method for glycosylation structural characterizations of a monoclonal antibody (mAb) using a newly developed quadrupole ion-mobility time-of-flight (ESI-Q-IM-TOF) mass spectrometer. Using this method, high-resolution mass spectra were acquired to produce the overall glycosylation profile of the mAb. Additionally, the light and heavy chains from the reduced antibody were separated in the gas phase by the ion mobility functionality of the instrument, allowing accurate mass measurement of each subunit. Furthermore, the glycan sequences, as well as the glycosylation site, were determined by a two-step sequential fragmentation process using the unique dual-collision-cell design of the instrument, thus providing detailed characterizations of the glycan structures.

A retention prediction model was developed for peptides separated in reversed-phase chromatography. The model was utilized to identify and exclude the false positive (FP) peptide identifications obtained via database search. The selected database included human proteins, as well as decoy sequences of random proteins. The FP peptide detection rate was defined either as number of retention time outliers, or random decoy sequence identifications. The FP rate for various MASCOT scores was calculated. The peptides identified in one-dimensional (1D) and two-dimensional (2D) liquid chromatography/mass spectrometry (LC/MS) experiments were validated by prediction models. Multi-dimensional LC was based on two orthogonal reversed-phase chromatography modes; prediction models were successfully applied for data filtering in both separation dimensions.

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